BIOTROPICA 41(6): 721–729 2009 10.1111/j.1744-7429.2009.00529.

Mecardonia tenella (Plantaginaceae) Attracts Oil-, Perfume-, and Pollen-Gathering Bees in

Southern Brazil

Simone Caroline Cappellari1,4, Birgit Harter-Marques1,2, Pia Aumeier1,3, and Wolf Engels1
1
Zoologisches Institut, Eberhard-Karls Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany

2
Universidade do Extremo Sul Catarinense, Programa de Pós-Graduação em Ciências Ambientais, Av. Universitária, 1105, C. P. 3167, 88806-000
Criciúma, Brazil

3
AG Verhaltensbiologie und Didaktik der Biologie, Universität Bochum, Universitätsstr. 150, Gebäude NCDF 06/494, D-44780 Bochum, Germany

ABSTRACT
Floral characteristics often indicate the pollinators’ functional group visiting the plant and the pollination syndromes associated with them. This idea has been chal-
lenged in the past decades due to increasing evidence that most plants, including those exhibiting floral syndromes, are visited by large arrays of species that differ in
their effectiveness as pollinators. Our study focuses on Mecardonia tenella (Plantaginaceae) from the Araucaria forest of southern Brazil, which exhibits characteristics
of the oil flower pollination syndrome. However, it is visited by three types of functional groups of bees: male orchid bees, oil-collecting bees, and pollen-seeking bees.
The relative contribution of each functional group to the plant’s reproductive success was estimated based on their pollen load, visitation frequency, and morphology.
We assessed resources, phenology, and breeding system of M. tenella. Our results indicate that flowers lack nectar, but volatiles, lipids, and pollen are resources that can
be gathered by visitors. This combination of floral traits and visitors’ assemblage makes M. tenella a challenge to the concept of pollination syndromes. Our findings
indicate that the current interactions may not reflect the circumstances under which some floral traits of this plant were selected.

Abstract in Portuguese is available at http://www.blackwell-synergy.com/loi/btp

Key words: Araucaria forest; Euglossa mandibularis; fatty acids; functional groups; GC–MS; oil flowers; orchid bees; pollination syndromes.

THE CONCEPT OF POLLINATION SYNDROMES has been a central topic in
the study of pollination ecology and evolution of plant–pollinator
interactions (Fenster et al. 2004). This term has been proposed in
recognition of convergence in floral traits (e.g., type of rewards,
morphology, floral advertisement) displayed by unrelated taxa of
angiosperms, which indicate the mode of pollination by the same
‘functional groups’ (Faegri & van der Pijl 1971, Fenster et al.
2004). Similar selective pressures imposed by particular functional
groups of pollinators are thought to shape these sets of convergent
floral characteristics because different species within each functional
group have similar sizes, nutritional needs, flower handling, sensory
systems (Fenster et al. 2004). For instance, the chemical composi-
tion, presentation, and quantity of rewards offered by flowers are
likely to be under selective pressures exerted by the nutritional and
ecological requirements of pollinators (Baker & Baker 1983, 1990;
Simpson & Neff 1983; Gerlach & Schill 1991; Petanidou 2005).
Good examples of functional group limitation imposed by specific
characteristics of rewards’ chemical composition are the ‘perfume
flower’ and the ‘oil flower’ pollination syndromes (Dodson 1975,
Simpson & Neff 1981, Vogel 1988, Eltz et al. 1999). The perfume
flower pollination syndrome is characterized by production of eth-
erical oils (fragrances) as rewards for pollinating male orchid bees
(Dressler 1982, Vogel 1990). In contrast, the offered floral rewards
Received 16 March 2008; revision accepted 22 February 2009.
4
Corresponding author; Current address: Section of Integrative Biology, The
University of Texas at Austin, 1 University Station A6700, Austin, TX 78712,
USA. e-mail: scappellari@mail.utexas.edu
r 2009 The Author(s)
Journal compilation r 2009 by The Association for Tropical Biology and Conservation
in the oil flower pollination syndrome are lipids (fatty acids) which
are collected by female oil-collecting bees for larval provision and
nest lining (Vogel 1974, Cane et al. 1983, Vinson et al. 1997). Both
orchid bees and oil-collecting bees have behavioral and morpho-
logical adaptations indicating evolutionarily (sensu Armbruster
2006) specialization for these pollination syndromes (Dodson
1975, Neff & Simpson 1981, Dressler 1982, Buchmann 1987).
Although these characteristics of plant–pollinator interactions
indicate the reciprocal effect of interacting species, effective polli-
nation can also be achieved by species of other functional groups
(Fenster & Dudash 2001, Mayfield et al. 2001, Valdivia & Niem-
eyer 2006). This happens because several pollinators are able to
perceive advertisement and explore rewards that might be directed
toward others (Waser et al. 1996). Furthermore, plant–pollinator
communities are subject to environmental influences that affect
plant and pollinator populations, such as introduction of exotic
species, climatic variation, and habitat change which can lead to
variation in strength of interactions as well to formation of new
associations (Roubik 1978, Pettersson 1991, Waser et al. 1996,
Gomez & Zamora 1999, Fenster & Dudash 2001, Price et al.
2005, Sargent & Vamosi 2008).
We here report on a nectarless member of the Plantaginaceae,
Mecardonia tenella (Chamisso & Schlechtendal) Pennell, which is
visited by three functional groups of bees (oil-, perfume-, and pol-
len-collecting) in a transition zone between the Araucaria forest and
the Atlantic rainforest in southern Brazil. The reproductive and
pollination systems of this species have not been previously studied
and close examination of floral traits reveals similarities with species
721
722 Cappellari, Harter-Marques, Aumeier, and Engels
that have the oil flower pollination syndrome. In addition, oil-
collecting bees occasionally do visit the flowers. However, male
orchid bees of Euglossa (Euglossella) mandibularis Friese and social
worker bees are the most frequent visitors. We assess pollen carry
over, visitation frequency, and chemo-ecological characteristics
of plant–pollinator relationships involving the three functional
groups, and discuss the relative importance of each to the repro-
ductive success of M. tenella. A survey of visitor spectra is provided.
Emphasis was given to understanding the novel interaction between
male orchid bees and flowers of M. tenella.
METHODS
STUDY SITE.—Fieldwork was conducted from October 2001 to Jan-
uary 2002 and from November 2003 to January 2004, at the Forest
Reserve Pró-Mata (FRPM) in the State of Rio Grande do Sul (RS)
in southern Brazil (29130 0 0500 S, 50110 0 0000 W). This 5000 ha area
is characterized by patches of Araucaria angustifolia (Bertol.)
Kuntze forest, deciduous forest, shrub vegetation, and grasslands
(Bertoletti & Teixeira 1997). Part of the area had been converted to
pasture before it became a forest reserve in 1993 (Bertoletti &
Teixeira 1997). FRPM is located at the edge of the Araucaria
plateau adjacent to the remaining strip of Atlantic rain forest at a
distance of ca 50 km from the southeastern Brazilian coast. Annual
precipitation is 2500 mm and the altitude of this area is
600–1000 m asl (Bertoletti & Teixeira 1997).
FLORAL MORPHOLOGY.—Mecardonia tenella is a herb, 8–15 cm in
height, which occurs in southern Brazil (states of Paraná, Santa
Catarina, and Rio Grande do Sul [RS]), northeastern Argentina,
western Paraguay, and Uruguay (Rossow 1987). It has perfect flow-
ers of 1–1.5 cm diam. with yellow zygomorphic corollas of four
united petals, one of which is larger and referred to as the lip (Fig.
1A, B; Rossow 1987). Two pairs of didynamous stamens are adnate
to the corolla tube, with one pair of anthers above and another pair
below the stigma (Rossow 1987). To determine if M. tenella has a
nectar disk, we examined 10 flowers under  20 magnification.
TRICHOMES.—We examined the trichomes found on petals under
 20 and  40 magnification and compared their shape and size
with those described as ‘trichome-elaiophores’ described by Vogel
(1974). Given that one oil-collecting bee species had been previ-
ously recorded on M. tenella (Harter 1999), the surface of petals
was extracted and analyzed for the occurrence of characteristic fatty
acids found in the fatty oils of oil flowers (see also ‘Chemical anal-
ysis’ in this section).
OSMOPHORES.—To determine if M. tenella produces volatiles that
attract male orchid bees, we carefully separated petals and sepals of
40 flowers from their reproductive parts, and tested them for the
occurrence of glands that emit scents, so called ‘osmophores’. We
used neutral red (1:1000) as a vital staining solution (Hess 1983).
Transverse histological cuts of the stained parts were made right af-
ter testing; results were evaluated using a light microscope at  40
magnification and compared with the morphology of osmophores
described in Vogel (1990).
PHENOLOGY AND BREEDING SYSTEM.—At the study site, populations
of M. tenella occur in open grasslands, building flower patches cov-
ering several square meters of rocky ground. For anthesis studies, a
total of 538 flowers were marked individually and monitored three
times a day for 8 consecutive days. From those, 128 flowers were
examined regarding pollen presentation on the long and short an-
thers, and stigma receptivity during anthesis. These data allow us to
see if the reproductive organs were synchronized to allow self-polli-
nation. Another 40 buds were marked and covered with a net to pre-
vent contact with visitors to test for autogamy by spontaneous self-
pollination. Fruits resulting from this test were collected, opened,
and seeds per fruit were counted. For the control treatment, we
marked 40 buds that were left uncovered and monitored for fruit
production. Tests for geitonogamy and xenogamy were unsuccessful.
Therefore, meaningful results are restricted to autogamy.
CHEMICAL ANALYSIS
HEADSPACE SAMPLING.—To detect the presence of volatiles on other
parts of M. tenella flowers that could potentially be responsible for
the attraction of male orchid bees, we extracted ten sepals using
closed-loop headspace equipment with a membrane pump for 12 h
followed by extraction with dichloromethane (W. Francke, pers.
comm.).
GAS-CHROMATOGRAPHIC/MASS-SPECTROMETRIC (GC–MS) ANALYSES.—
To characterize the major compounds of the liquid inside the tric-
homes of M. tenella we extracted 50 corollas. Each sample was
washed in 2 mL of dichloromethane and four consecutive extraction
periods of 1, 5, 10 and 30 min were applied to each sample (W.
Francke, pers. comm.).
The same quantity of dichloromethane and extraction times
were used to extract bee body parts in order to find out if the com-
pounds present in the flowers of M. tenella were actively collected
and stored by male orchid bees. We separately extracted four body
parts of orchid bee males: heads, fore-, mid-, and hind-legs. All
samples were subject to cold evaporation, concentration, partial hy-
drolysis and methylation using a protocol by T. Härtner (Univer-
sity of Tübingen, Germany). Samples were analyzed by GC–MS.
GC–MS analyses were done using a Shimadzu GC-17A with
MS QP5000, Column Optima 5 (methyl silicon, nonpolar, Ø
0.25 mm, 15 m). The temperature program used was 901C for
5 min, 90–2001C with stepwise increase of 201C/min, 200–3001C
at 41C/min finalizing with 3001C for 2 min (isotherm). Injector
temperature was 3201C, detector temperature 3001C, split 1:4 and
total flow of 4 mL/min.
Compounds were identified by a combination of retention
time (RT), comparing spectra from the substance library Bench
Top/BMP (Palisade) using a hydrocarbon standard mix, and by
comparison with published spectra of oil flowers (Vogel 1974,
Simpson et al. 1977). All analyses of chemical compounds reported
in this study are qualitative.
 Pollination Ecology of Mecardonia tenella 723

FIGURE 1. (A) Mecardonia tenella flower, note the occurrence of trichomes on the lip; (B) Detail of petal lip with trichomes; (C) Sepals before (left) and after (right)
staining for osmophores; (D) Male Euglossa mandibularis collecting volatiles, and perhaps lipids, on flower; (E) Male collecting volatiles on sepals after corolla broke
off (note corolla laying on the ground, at the lower left side); (F) Dead male specimen of E. mandibularis after collection on M. tenella with orchid pollinia attached to
dorsum of bee’s prothorax.
724 Cappellari, Harter-Marques, Aumeier, and Engels
VISITORS DIVERSITY, VISITATION FREQUENCY AND POLLEN ANALYSES.—
To determine the spectra and frequency of visitation to M. tenella
flowers, we collected all insects that visited individual flowers of two
patches of flowers within a total area of 100 m2 (ca 500 flowers) for
30 min three times a day for 2 consecutive days once a week from
November to January during our second field season (2003–2004).
A total of 36 h of observations distributed over 72 censuses were
recorded. Visitation frequency (hereafter referred to as ‘F’) of each
visiting species per hour was calculated as the number of individuals
collected per species divided by the total number of hours of cen-
suses recorded. Bees were identified by B. Harter-Marques accord-
ing to Michener (2000) and Silveira et al. (2002), and by
comparisons with local collections. All specimens were deposited
in the LPB collection of the PUCRS Museu de Ciências e
Tecnologia, Porto Alegre, Brazil.
Loose pollen grains available on the body of visitors after vis-
iting M. tenella were sampled with small blocks of stained glycerin
jelly and mounted on microscope slides (Hess 1983). Pollen loads
from bees’ collecting structures (e.g., corbicula, scopae) were not
included. Pollen slides were made for each bee species with samples
from at least four individuals collected on flowers. The percentage
of pollen from M. tenella found on a bee body was evaluated count-
ing 300 pollen grains of each pollen slide and the mean percent
of pollen from M. tenella per bee species was calculated (after
Schlindwein 1995).
ORCHID BEES AT FLOWERS.—Euglossa mandibularis is a widespread
species of the tribe Euglossini. In Brazil, this species occurs from
southeastern to southern (Wittmann et al. 1988, Nemésio & Silve-
ira 2007), and males are associated with perfume flowers of
Cyphomandra (Solanaceae; Soares et al. 1989, Sazima et al. 1993).
During the study periods, we continuously observed the foraging
behavior of male orchid bees on the flowers of M. tenella. A total of
85 individual observations were documented between 0730 h and
1830 h. We measured temperature and recorded the time of day
when males were seen foraging on flowers. To test the hypothesis
that male E. mandibularis actively collect volatiles on the flowers,
we recorded their behaviors: (1) when approaching a flower patch;
(2) once they landed on the flower; and (3) after they had left the
flower. Evaluation of male behavior on flowers as scent collecting or
feeding was based on the description of Dressler (1982), and pre-
vious observations of male E. mandibularis collecting scents on
b-ionone baits (S. C. Cappellari, pers. obs.). Males were only col-
lected from the population on days when visitors were surveyed to
determine the visitor’s spectra on the flowers.
RESULTS
MORPHOLOGY, PHENOLOGY AND BREEDING SYSTEM.—The flowers of
M. tenella have a narrow corolla entrance, which is about 0.5 cm
wide with four united petals. Flowers are bent, the anthers and
stigma are not exserted, and visitors use the lip as support structure
to enter the flower. Anthers are positioned on the inner lateral and
ventral side of the corolla, thus dorsolateral and ventral parts of vis-
itor’s body contact anthers inside the corolla. The stigma is bent
and located in the middle of the corolla tube. Examination of the
flower under high magnification indicates that this species lacks a
nectar disk. All petals display small trichomes on the basis of the
corolla and large trichomes are located on the lip (Fig. 1A, B). Tests
for detection of osmophores on corolla and calyx yielded positive
results. Each petal has one to two osmophore regions in an area
where the corolla petals become fused. The osmophores found on
sepals were smaller, more numerous (more than 40/sepal) and
evenly distributed (Fig. 1C). These osmophores are characterized
by a ring of cells with a central opening.
At our study site, M. tenella blooms from November through
March. The mean flower longevity is 4.1 d (  1.0 SD, N = 538).
Under natural conditions, fruit capsules averaged 150 seeds/fruit
(  7.4 SD, N = 10). Even though 24 percent (N = 31) of the 128
flowers tested had long anthers dehiscing concurrently with stigma
receptivity, which would allow for selfing, only 7.5 percent (N = 3)
of the flowers tested for autogamy by spontaneous self-pollination
produced fruits with an average of 74 seeds/fruit (  66.7 SD) while
the control (N = 40) yielded 13 fruits.
CHEMICAL ANALYSES.—In GC–MS analyses of flower extracts, we
identified alkanes, methyl-alkanes, alkenes, fatty acid esters, and
free fatty acids among other compounds (Table S1). Infrared
analysis confirmed the presence of carboxyl groups, characteristic
of organic acids, in the compounds of the floral extracts. In addi-
tion, flower extracts had a high concentration of short-chained
substances appearing in the first 6 min of the RT indicating
the presence of volatiles some of which could not be properly iden-
tified by GC–MS analysis. Small quantities of the monoterpene
Azulen were detected in headspace sepals extracts analyzed with
GC–MS.
The results of GC–MS analysis of the extracted bee parts
showed that traces of M. tenella compounds could be detected in
extracts of E. mandibularis’s fore, mid and hind legs (Table S1).
Among the compounds were several alkanes, alkenes and fatty acids
(Table S1). No flower compounds could be detected in head
extracts of E. mandibularis.
FLOWER VISITORS AND POLLEN ANALYSES.—A total of 217 bees of 23
species belonging into four families were recorded visiting flowers
of M. tenella. Throughout the observation period, workers of Plebe-
ia emerina (N = 63) were the most frequent visitors (F = 1.75;
Table 1). Males of E. mandibularis were the second most abundant
visitors (F = 1.64, N = 59). If only the 6-wk period (November–
December) of their occurrence at the study site was considered,
then E. mandibularis had the highest frequency of visitation to
flowers (F = 3.28, N = 59; Table 1). One female and three males of
the oil-collecting bee Chalepogenus betinae (Apidae: Tapinotaspi-
dini) were collected on the flowers (F = 0.11, N = 4).
The analysis of P. emerina individual workers (N = 20)
showed an average of 80.8 percent of pollen grains from M. tenella
(Table 1). Pollen analyses of E. mandibularis (N = 4) indicate
an average 82 percent of pollen grains from M. tenella. Pollen
quantity found on specimens of C. betinae was insufficient for an-
alyses. Other flower visitors carrying pollen of M. tenella were
 Pollination Ecology of Mecardonia tenella 725

TABLE 1. List of bee species visiting flowers of Mecardonia tenella recorded over 72 censuses of 30 min each during the period of November 2003 to January 2004. Reported
are counts for each species (N) subdivided into female (,) and male (<) counts; proportion was calculated as the total number of individuals per species divided by
total number of all bees collected; frequency of visitors per hour per patch of flowers was calculated as number of individuals collected per species divided by total
number of hours of censuses recorded; % pollen is the average percentage of pollen from M. tenella on the body of visitors available for pollination; type of resource
indicates the floral resources collected by bees on flowers as follows: P = pollen, V = volatiles, and L = lipids. No information is indicated by ‘‘–.’’

Family Species , < N Proportion (%) Frequency % Pollen Resource

Andrenidae Anthrenoides DUCKE sp. 1 7 2 9 4.15 0.25 – –

Anthrenoides DUCKE sp. 2 2 2 4 1.84 0.11 – –
Psaenythia collaris SCHROTTKY 7 0 7 3.23 0.19 – –
Psaenythia bergi HOLMBERG 1 0 1 0.46 0.03 –
Apidae Apis mellifera LINNAEUS 15 0 15 6.91 0.42 81 P
Ceratina muelleri FRIESE 3 0 3 1.38 0.08 – –
Ceratina hyemalis MOURE 1 0 1 0.46 0.03 – –
Ceratina LATREILLE sp. 2 7 0 7 3.23 0.19 70.6 –
Chalepogenus betinae URBAN 1 3 4 1.84 0.11 – –
Euglossa mandibularis FRIESE 0 59 59 27.2 1.64 82 V, L?
Plebeia emerina FRIESE 63 0 63 29.0 1.75 80.8 P
Plebeia remota HOLMBERG 2 0 2 0.92 0.06 – –
Schwarziana quadripunctata LEPELETIER 2 0 2 0.92 0.06 –
Colletidae Hexantheda missionica OGLOBIN 1 0 1 0.46 0.03 –
Halictidae Augochlora semiramis SCHROTTKY 7 0 7 3.23 0.19 – –
Augochlora SMITH sp. 1 1 0 1 0.46 0.03 – –
Augochloropsis euterpe HOLMBERG 7 0 7 3.23 0.19 76.8 –
Augochloropsis COCKERELL sp. 3 17 0 17 7.83 0.47 83.3 –
Dialictus ROBERTSON sp. 1 1 0 1 0.46 0.03 – –
Dialictus ROBERTSON sp. 2 1 0 1 0.46 0.03 – –
Dialictus ROBERTSON sp. 3 0 1 1 0.46 0.03 – –
Paroxystoglossa brachycera MOURE 2 0 2 0.92 0.06 – –
Pseudagapostemon fluminensis SCHROTTKY 2 0 2 0.92 0.06 – –
Total 150 67 217 100

(N = number of bees used for pollen samples, x = average percent-
age of pollen on the bee body): Apis mellifera L. (N = 14, x = 81%),
Augochloropsis euterpene (N = 7, x = 76.8%), Augochloropsis sp. 3
(N = 15, x = 83.3%), and Ceratina sp. 2 (N = 5, x = 70.6%). The
type of resource collected by each species was recorded for the most
frequent visitors only (Table 1).
BEHAVIOR OF ORCHID BEES AT FLOWERS.—During the study period,
male E. mandibularis visited M. tenella flowers between 1000 h and
1500 h when temperatures were 4 251C. The males were observed
manipulating the petals and sepals of flowers (Fig. 1D, E) in a
manner similar to the way they collect chemical baits of b-ionone
(S. C. Cappellari, pers. obs.) and other sources of volatiles (Dressler
1982). While flying over the floral carpets, males hovered above the
flowers until a particular flower was selected. After alighting on the
corolla, they held the petals with their middle legs and stuck their
forelegs into the flowers (Fig. 1D). They then scratched the petals
for up to 3 min. While scratching the petals, males moved clockwise
or counterclockwise on the flower touching the reproductive parts
with the forelegs. When finished collecting on one flower, they
hovered while transferring the gathered substances from the forelegs
into the storage structures located on their hind legs. Males consis-
tently visited several flowers in a patch before leaving for another
area. Occasionally, males with orchid pollinia attached to the dor-
sum of their thoraces were observed at their arrival on flower
patches (Fig. 1F). None of the pollinia collected from the bees body
matched the pollinia of orchid species known for the study area.
Females of E. mandibularis were never observed on M. tenella dur-
ing the study.
After an E. mandibularis visit, the floral trichomes on the co-
rolla lip were visibly crushed and the corolla so damaged that it
usually abscised within a day. Sometimes males continued to
scratch on the sepals after the flowers had fallen off (Fig. 1E). Other
males were observed opening buds even though recently opened
flowers could be found in close proximity. While foraging, the
males kept their tongues folded and appressed to their bodies; no
feeding behavior was exhibited. When observed through a light mi-
croscope, male E. mandibularis caught on M. tenella flowers showed
traces of a shiny, greasy substance, and even pieces of trichomes, on
their hind legs.
726 Cappellari, Harter-Marques, Aumeier, and Engels
DISCUSSION
The floral characteristics of M. tenella cannot be clearly associated
with one particular functional group of bees that visits it. Conse-
quently, the classification of this species into a pollination syn-
drome is difficult even though the flowers display traits that are
characteristic of oil flowers, like the floral trichomes, presence
of free-fatty acids, and visits by oil-collecting bees (Vogel 1974,
Buchmann 1987). Although the oil flower pollination syndrome
has never been proposed for any species of Mecardonia (Vogel &
Cocucci 1995), Gratiolae is the sister group (Olmstead et al. 2001,
Albach et al. 2005) to the clade that contains the genera of neo-
tropical oil flowers Angelonia, Basistemon, and Monttea (Simpson
et al. 1990, Vogel & Machado 1991, Vogel & Cocucci 1995). This
supports the idea that M. tenella may have retained a possible
ancestral state for oil production. Chalepogenus (Lanthanomelissa)
betinae has been reported as an oil-collecting bee of Iridaceae,
Scrophulariaceae, and Solanaceae species (Vogel 1974, Cocucci &
Vogel 2001, Truylio et al. 2002). Interestingly, mostly males of this
species were caught on M. tenella (N = 3; Table 1) in this and past
surveys (Harter 1999). Males of other oil-collecting bees, mainly
Tapinotaspidini, have been recorded on oil flowers (Singer &
Cocucci 1999), some of them carrying oils in their tarsal scopae
(Cocucci 1991, Vogel & Machado 1991). However, the purpose of
oil-collecting by males is not clear as they do not participate in nest
maintenance or larval provisioning (Rozen et al. 2006). On the
other hand, the presence of volatiles on floral extracts and their col-
lection by male orchid bees are weaker indicators for the classifica-
tion of this species as a perfume flower. First, it is not entirely clear
that the fragrances produced in the osmophores are rewards to poll-
inators. Histological analysis of the osmophores found on flowers
would be a necessary first step to determine if they are morphologi-
cally similar to ordinary scent glands or to those described for other
perfume flowers (Vogel 1990). Second, the use of M. tenella as a
source of volatiles by male orchid bees may be purely opportunistic as
the presence of volatiles on flowers may be a byproduct of floral de-
fense against herbivores or advertisement to pollinators. In fact, males
of some orchid bee species are known to have different sources of
volatiles over their distribution range (Ackerman 1989, Ramı́rez et al.
2002, Roubik & Hanson 2004) and can even switch to alternative
fragrances if the usually visited host plants are not available (Roberts
et al. 1982, Whitten et al. 1993, Pemberton & Wheeler 2006). For
instance, males of Euglossa viridissima collect volatiles from the leaves
of aromatic plants such as basil and allspice in southern Florida where
this orchid bee, native to Central America, has been introduced but
no perfume orchids occur (Pemberton & Wheeler 2006).
Although the collecting behavior of male E. mandibularis on
sepals after the dehiscence of the corolla can be explained by the
presence of volatiles on this plant tissue, it is not entirely clear if
fatty acids found on males’ hind leg extracts are collected on pur-
pose or as a by-product of the collection of volatiles from the petals’
osmophores. The former scenario, although less parsimonious,
considers the idea that fatty oils from oil flowers could be used as
substitute for the labial secretions which are normally produced by
male orchid bees as a nonpolar solvent that reduces the loss of fra-
grances through evaporation. Labial glands secretions are composed
by fatty acid derivates and orchid bees mix them with the volatiles
they collect from perfume sources (Whitten et al. 1989). This be-
havioral ‘processing’ of scents shares the same principle of ‘enfleu-
rage,’ a method used in perfumery which uses pig lard as a solid
solvent for perfumes (Whitten et al. 1989, Eltz et al. 2007). Recent
studies have shown that at least some species of orchid bees have a
quite efficient mechanism of recycling these lipids indicating that
these substances are costly to produce (Eltz et al. 2007). Our results
show that some of the compounds found in samples of M. tenella
are also present in labial gland secretions of other orchid bees (Wil-
liams & Whitten 1983; Table S1). Thus, we hypothesize that
M. tenella flowers may have a fortuitous combination of floral com-
pounds, volatile attractants, and nonvolatile lipids, both of which
can be used by male orchid bees. The use of external sources of lip-
ids by male orchid bees is plausible if we consider that some bees are
known to use exogenous (floral) sources as nest linings (Cane et al.
1983) instead of endogenous secretions of the Dufour’s gland like
most solitary, ground nesting bees. To test the hypothesis, the size
of labial glands of E. mandibularis males known to use endogenous
lipid sources in regions within and without the occurrence range of
M. tenella should be compared.
Our breeding system experiments as well as our observations
of visitors indicate that pollination of M. tenella is mostly mediated
by biotic vectors (bees). While the fruits formed by the bagged
flowers could be due to apomixis, the fact that only 3 of the
40 covered flowers produced fruits strongly suggests that the plants
are self fertile and can occasionally produce fruits by selfing. How-
ever, the average of seeds per fruit produced by covered flowers was
less than half of the observed seed production per fruit under nat-
ural conditions. Based on our results, its main pollinator is most
likely to be a generalist, pollen-gathering stingless bee (P. emerina).
This conclusion is based on the assumption that visitation fre-
quency, percentage of pollen carry over, and contact with floral re-
productive parts are indicators of pollination effectiveness (Spears
1983; Schemske & Horvitz 1984; Herrera 1987, 1989). Although
we did not measure visitation frequency of P. emerina workers dur-
ing the entire blooming season of M. tenella, individuals of this
species have been recorded at our study site from October to April
(Harter 1999). In addition, other pollen-gathering bees (A. mellif-
era and Augochloropsis sp. 3) were also frequent visitors and poten-
tially have a positive effect as pollinators. Interactions with oil-
collecting bees (C. betinae and Paratetrapedia pygmaea) in this
(Cappellari 2003) and past surveys (Harter 1999) of visitors of M.
tenella indicate that this functional group plays a minor role in pol-
lination of this flower, if any, due to the low visitation frequency
and pollen carry over (Table 1). Male orchid bees are likely to con-
tribute to pollination even though these large bees often damage the
small corolla, and their visits to M. tenella occur only during a short
period (6 wk) of its blooming season (20 wk). However, long-dis-
tance flyers such as orchid bees ( Janzen 1971, Kroodsma 1975)
could mediate cross-pollination between populations of M. tenella
across the Araucaria plateau.
The current interactions of M. tenella and its visitors do not
necessarily reflect the circumstances under which it has evolved.

The lack of similarities between the three functional groups of bees
that visit its flowers and the floral characteristics can be explained by
several factors that affect intensity of plant-pollinator interactions at
the species and community levels. For instance, abiotic factors are
known to affect interactions (e.g., shifts in phenology due to climate
variation) by causing fluctuations in plant and pollinator popula-
tions (Wolfe & Barrett 1988, Armbruster 2006, Alarcon et al.
2008). In addition, variation in community composition, species
abundances, and resource availability are factors that can lead to
formation of new interactions, change in the intensity of pre-exis-
tent interactions as well as promote opportunistic interactions
(Herrera 1988, Price et al. 2005, Lázaro et al. 2008, Wang et al.
2008). For instance, the low number of oil-collecting bees recorded
on these flowers could be a result of nesting site loss through grazing
in the area until 1991 (Bertoletti & Teixeira 1997), which perhaps
led to a decrease in populations of ground nesting oil-collecting
bees in the past years. Furthermore, habitat fragmentation of the
Atlantic forest nearby (Ministério do Meio Ambiente 1999) which
bears most of the perfume sources used by orchid bees in this region
(Rambo 1956, 1960; Wittmann et al. 1988) could have led to the
use of alternative sources from adjacent areas. Consequently, M.
tenella and other plants from the Araucaria forest adjacent to the
Atlantic rainforest could have become fragrance hosts used by or-
chid bees in certain periods of the year. Orchid bees are known
to cross large and steep areas to forage on plants that occur at
low densities and/or outside of their primary habitat (rain forest)
( Janzen 1971, Dressler 1982, Janzen et al. 1982, Ackerman &
Montalvo 1985, Roubik 1989, Tonhasca et al. 2003). However, all
these factors that may be responsible for variation in the interaction
are not detectable by short-term studies that focus on a single spe-
cies of plant. Therefore, long-term studies would be necessary to
determine the relative importance of interactions with different
functional groups of bees for the evolution of floral characteristics
in M. tenella. In addition, comparative studies at the community
level in areas where these species overlap would help clarifying the
pollination system of this plant and explain if the interactions ob-
served between M. tenella and the three different functional groups
of pollinators that visit it are dependent on local community-
context.
ACKNOWLEDGMENTS
We thank: B. Blochtein and the Instituto do Meio Ambiente
PUCRS for logistical support; B. Truylio for support with field-
work; A. A. Souto and M. R. Pires, PUCRS, for their help with the
preliminary analyses; T. Härtner, G. J. Nicholson, and P. Rosenk-
ranz for invaluable help with the GC–MS analyses; and B. Klein for
formatting the figures. SCC thanks D. Wittmann and A. Garófalo
for inspiring discussions about Euglossine bees. We are grateful to
B. B. Simpson, J. L. Neff, C. Rabeling, and two anonymous re-
viewers for comments on earlier versions, which greatly improved
this manuscript. This study was supported by the Brazilian
Research Council (CNPq) Grant 479138/01-0 to BHM and
SCC. The research was part of the projects Araucaria forest
(BMBF-DLR-IB) and Araucaria plateau (FAPERGS-DLR).
Pollination Ecology of Mecardonia tenella 727
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
TABLE S1. Chemical compounds found in Mecardonia tenella and
in Euglossa mandibularis samples analyzed by GC–MS.
Please note: Wiley-Blackwell are not responsible for the content or
functionality of any supporting materials supplied by the authors.
Any queries (other than missing material) should be directed to the
corresponding author for the article.
LITERATURE CITED
ACKERMAN, J. D. 1989. Geographic and seasonal variation in fragrance choices
and preferences of male euglossine bees. Biotropica 21: 340–347.
ACKERMAN, J. D., AND A. M. MONTALVO. 1985. Longevity of euglossine bees.
Biotropica 17: 79–81.
ALARCON, R., N. M. WASER, AND J. OLLERTON. 2008. Year-to-year variation in
the topology of a plant-pollinator interaction network. Oikos. 117:
1796–1807.
ALBACH, D. C., H. M. MEUDT, AND B. OXELMAN. 2005. Piecing together the new
Plantaginaceae. Am. J. Bot. 92: 297–315.
ARMBRUSTER, W. S. 2006. Evolutionary and ecological aspects of specialized
pollination: Views from the Arctic to the Tropics. In N. M. Waser and
J. Ollerton (Eds.): Plant–pollinator interactions: From specialization
to generalization. pp. 260–282. University of Chicago Press, Chicago,
Illinois.
BAKER, H. G., AND I. BAKER. 1983. Floral nectar sugar constituents in relation to
pollinator Type. In C. E. Jones and R. J. Little (Eds.): Handbook of
experimental pollination ecology. pp. 117–141. Van Nostrand-Rein-
hold, New York, New York.
BAKER, H. G., AND I. BAKER. 1990. The predictive value of nectar chemistry to
the recognition of pollinator types. Isr. J. Botan. 39: 157–166.
BERTOLETTI, J. J., AND M. B. TEIXEIRA. 1997. Centro de Pesquisas e Conservação
da Natureza Pró-Mata: Plano de Trabalho para a Elaboração do Plano
de Manejo. Divulgações do Museu de Ciências e Tecnologia (UBEA/
PUCRS) Porto Alegre 3: 3–27.
BUCHMANN, S. 1987. The ecology of oil flowers and their bees. Annu. Rev. Ecol.
Syst. 18: 343–369.
CANE, J. H., G. C. EICKWORT, F. R. WESLEY, AND J. SPIELHOLZ. 1983. Foraging,
grooming and mate-seeking behaviors of Macropis nuda and use of
Lysimachia ciliata (Primulaceae) oils in larval provision and cell linings.
Am. Midl. Nat. 110: 257–264.
CAPPELLARI, S. C. 2003. Blüten von Mecardonia tenella (Scrophulariaceae) als
alternative Duftstoff-Trachtquelle für Männchen von Euglossa mandib-
ularis (Apidae: Euglossini) – Fallstudie Pró-Mata, Rio Grande do Sul,
Brasilien. MSc Dissertation. Eberhard-Karls Universität Tübingen,
Germany.
COCUCCI, A. A. 1991. Pollination biology of Nierembergia. Pl. Syst. Evol. 174:
17–35.
COCUCCI, A. A., AND S. VOGEL. 2001. Oil-producing flowers of Sisyrinchium
species (Iridaceae) and their pollinators in southern South America.
Flora 196: 26–46.
DODSON, C. H. 1975. Coevolution of orchids and bees. In L. E. Gilbert and
P. H. Raven (Eds.): Coevolution of animals and plants. pp. 91–99.
University of Texas Press, Austin, Texas.
DRESSLER, R. L. 1982. Biology of the orchid bees (Euglossini). Annu. Rev. Ecol.
Syst. 13: 373–394.
ELTZ, T., W. M. WHITTEN, D. W. ROUBIK, AND K. E. LINSENMAIR. 1999.
Fragrance collection, storage, and accumulation by individual male
orchid bees. J. Chem. Ecol. 25: 157–176.
728 Cappellari, Harter-Marques, Aumeier, and Engels
ELTZ, T., Y. ZIMMERMANN, J. HAFTMANN, R. TWELE, W. FRANCKE, J. J. G. QUE-
ZADA-EUAN, AND K. LUNAU. 2007. Enfleurage, lipid recycling and the
origin of perfume collection in orchid bees. Proc. R. Soc. B 274:
2843–2848.
FAEGRI, K., AND L. VAN DER PIJL. 1971. The principles of pollination ecology.
Pergamon Press, Oxford, UK.
FENSTER, C. B., W. S. ARMBRUSTER, P. WILSON, M. R. DUDASH, AND J. D.
THOMSON. 2004. Pollination syndromes and floral specialization. Ann.
Rev. Ecol. Evol. Syst. 35: 375–403.
FENSTER, C. B., AND M. R. DUDASH. 2001. Spatiotemporal variation in the role
of hummingbirds as pollinators of Silene virginica (Caryophyllaceae).
Ecology 82: 844–851.
GERLACH, G., AND R. SCHILL. 1991. Composition of orchid scents attracting
euglossine bees. Bot. Acta 104: 379–391.
GOMEZ, J. M., AND R. ZAMORA. 1999. Generalization vs. specialization in the
pollination system of Hormathophylla spinosa (Cruciferae). Ecology 80:
796–805.
HARTER, B. 1999. Bienen und ihre Trachtpflanzen im Araukarien-Hochland
von Rio Grande do Sul, mit Fallstudien zur Bestäubung von Pi-
onierpflanzen. PhD Dissertation. Eberhard-Karls Universität Tübingen,
Germany.
HERRERA, C. M. 1987. Components of pollinator ‘‘quality’’: Comparative anal-
ysis of a diverse insect assemblage. Oikos 50: 79–90.
HERRERA, C. M. 1988. Variation in mutualisms: The spatiotemporal mosaic of
a pollinator assemblage. Biological Journal of the Linnean Society 35:
95–125.
HERRERA, C. M. 1989. Pollinator abundance, morphology, and flower visitation
rate: Analysis of the ‘‘quantity’’ component in a plant-pollinator system.
Oecology 80: 241–248.
HESS, D. 1983. Die Blüte – eine Einführung in Struktur und Funktion, Ökologie
und Evolution der Blüten. Eugen-Ulmer-Verlag, Stuttgart, Germany.
JANZEN, D. H. 1971. Euglossine bees as long-distance pollinators of tropical
plants. Science 171: 203–205.
JANZEN, D. H., P. J. DEVRIES, M. L. HIGGINS, AND L. S. KIMSEY. 1982. Seasonal
and site variation in Costa Rican euglossine bees at chemical baits in
lowland deciduous and evergreen forests. Ecology 63: 66–74.
KROODSMA, D. E. 1975. Flight distances of male euglossine bees in orchid pol-
lination. Biotropica 7: 71–72.
LÁZARO, A., S. J. HEDLAND, AND Ø. TOTLAND. 2008. The relationships between
floral traits and specificity of pollination systems in three Scandinavian
plant communities. Oecologia 157: 249–257.
MAYFIELD, M. M., N. M. WASER, AND M. V. PRICE. 2001. Exploring the ‘most
effective pollinator principle’ with complex flowers: Bumblebees and
Ipomopsis aggregata. Ann. Bot. 88: 591–596.
MICHENER, C. 2000. The bees of the World. John Hopkins University Press,
Baltimore, Maryland.
MINISTÉRIO DO MEIO AMBIENTE. 1999. Diretrizes para a polı́tica de conservação e
desenvolvimento sustentável da Mata Atlântica. Caderno no 13. Brası́lia,
Distrito Federal, Brazil.
NEFF, J. L., AND B. B. SIMPSON. 1981. Oil-collecting structures in the Ant-
hophoridae (Hymenoptera): Morphology, function, and use in systema-
tics. J. Kans. Entomol. Soc. 54: 95–123.
NEMÉSIO, A., AND F. A. SILVEIRA. 2007. Diversity and distribution of orchid
bees (Hymenoptera: Apidae) with a revised checklist of species. Neotr.
Entomol. 36: 874–888.
OLMSTEAD, R. G., C. W. PAMPHILIS, A. D. WOLFE, N. D. YOUNG, W. J. ELISONS,
AND P. A. REEVES. 2001. Disintegration of the Scrophulariaceae. Am.
J. Bot. 88: 348–361.
PEMBERTON, R. W., AND G. S. WHEELER. 2006. Orchid bees don’t need orchids:
Evidence from the naturalization of an orchid bee in Florida. Ecology
87: 1995–2001.
PETANIDOU, T. 2005. Sugars in Mediterranean floral nectars: An ecological and
evolutionary approach. J. Chem. Ecol. 31: 1065–1088.
PETTERSSON, M. W. 1991. Pollination by a guild of fluctuating moth population:
Options for unspecialization in Silene vulgaris. J. Ecol. 79: 591–604.
PRICE, M. V., N. M. WASER, R. E. IRWIN, D. R. CAMPBELL, AND A. K. BRODY.
2005. Temporal and spatial variation in pollination of a montane herb:
A seven year study. Ecology 86: 2106–2116.
RAMBO, B. 1956. Der Regendwald am oberen Uruguay. Sellowiana An. Bot. 7:
183–233.
RAMBO, B. 1960. Die Südgrenze des brasilianischen Regenwaldes. Pesquisas Bot.
8: 5–41.
RAMÍREZ, S., R. L. DRESSLER, AND M. OSPINA. 2002. Abejas euglosinas (Hymen-
optera: Apidae) de la región neotropical: Listado de especies con notas
sobre su biologı́a. Biota Colombiana 3: 7–118.
ROBERTS, D. R., W. D. ALECRIM, J. M. HELLER, S. R. EHRHARDT, AND J. B. LIMA.
1982. Male Eufriesia purpurata, a DDT-collecting euglossine bee in
Brazil. Nature 297: 62–63.
ROSSOW, R. A. 1987. Revisión del género Mecardonia (Scrophulariaceae). Can-
dollea (Buenos Aires) 42: 431–474.
ROUBIK, D. W. 1978. Competitive interactions between neotropical pollinators
and Africanized honey bees. Science 201: 1030–1032.
ROUBIK, D. W. 1989. Ecology and natural history of tropical bees. Cambridge
Univ. Press, New York, New York.
ROUBIK, D. W., AND P. E. HANSON. 2004. Orchid bees of tropical America:
biology and field guide. InBIO Press, Heredia, Costa Rica.
ROZEN, J. JR., G. A. R. MELO, A. J. C. AGUIAR, AND I. ALVES-DOS-SANTOS. 2006.
Nesting biologies and immature stages of the Tapinotaspidine Bee Gen-
era Monoeca and Lanthanomelissa and of their Osirine cleptoparasites
Protosiris and Parepeolus (Hymenoptera: Apidae: Apinae). Am. Mus.
Novitat. 3501: 1–60.
SARGENT, R. D., AND J. C. VAMOSI. 2008. The influence of canopy position,
pollinator syndrome, and region on evolutionary transitions in poll-
inator guild size. Int. J. Plant Sci. 169: 39–47.
SAZIMA, M., S. VOGEL, A. A. COCUCCI, AND G. HAUSNER. 1993. The perfume
flowers of Cyphomandra (Solanaceae) – Pollination by euglossine bees, bel-
lows mechanisms, osmophores and volatiles. Plant Syst. Evol. 187: 51–88.
SCHEMSKE, D. W., AND C. C. HORVITZ. 1984. Variation among floral visitors in
pollination ability: A precondition for mutualism specialization. Science
225: 519–521.
SCHLINDWEIN, C. 1995. Wildbienen und ihre Trachtpflanzen in einer
südbrasilianischen Buschlandschaft: Fallstudie Guaritas, Bestäubung
bei Kakteen und Loasaceen. PhD Dissertation, Eberhard-Karls Univer-
sität Tübingen, Germany.
SILVEIRA, F. A., G. A. R. MELO, AND E. A. B. ALMEIDA. 2002. Abelhas brasileiras:
sistemática e identificação. Ministério do Meio Ambiente, Fundação
Araucária, Belo Horizonte, Brazil.
SIMPSON, B. B., AND J. L. NEFF. 1981. Floral rewards: Alternatives to pollen and
nectar. Ann. Mo. Bot. Gard. 68: 301–322.
SIMPSON, B. B., AND J. L. NEFF. 1983. Evolution and diversity of floral rewards.
In C. E. Jones and R. J. Little (Eds.): Handbook of experimental polli-
nation ecology. pp. 277–293. Van Nostrand-Reinhold, New York,
New York.
SIMPSON, B. B., J. L. NEFF, AND G. DIERINGER. 1990. The production of floral
oils by Monttea (Scrophulariaceae) and the function of tarsal pads in
Centris bees. Plant Syst. Evol. 173: 209–222.
SIMPSON, B. B., J. L. NEFF, AND D. SEIGLER. 1977. Krameria, free fatty acids and
oil-collecting bees. Nature 267: 150–151.
SINGER, R. B., AND A. A. COCUCCI. 1999. Pollination mechanisms in four
sympatric southern Brazilian Epidendroideae orchids. Lindleyana 14:
47–56.
SOARES, A. A., L. A. D. O. CAMPOS, M. F. VIEIRA, AND G. A. R. D. MELO. 1989.
Relationship between Euglossa mandibularis FRIESE 1899 (Hymenopt-
era, Apidae, Euglossini) and Cyphomandra calycina (Solanaceae). Ciên-
cia e Cultura 41: 903–905.
SPEARS, E. E. JR. 1983. A direct measure of pollinator effectiveness. Oecologia
57: 196–199.
TONHASCA, A. J., G. S. ALBUQUERQUE, AND J. L. BLACKMER. 2003. Dispersal
of euglossine bees between fragments of the Brazilian Atlantic Forest.
J. Trop. Ecol. 19: 100–102.

TRUYLIO, B., B. HARTER-MARQUES, AND W. ENGELS. 2002. Biologia floral e
polinização de Sisyrinchium micranthum (Iridaceae) na região do Plan-
alto das Araucárias do Rio Grande do Sul, Brasil. Biociências 10: 11–24.
VALDIVIA, C. E., AND H. E. NIEMEYER. 2006. Do floral syndromes predict
specialisation in plant pollination systems? Assessment of diurnal and
nocturnal pollination of Escallonia myrtoidea. New Zeal. J. Bot. 44:
135–141.
VINSON, S. B., H. J. WILLIAMS, G. W. FRANKIE, AND G. SHRUM. 1997. Floral lipid
chemistry of Byrsonima crassifolia (Malpigheaceae) and a use of floral
lipids by Centris bees (Hymenoptera: Apidae). Biotropica 29: 76–83.
VOGEL, S. 1974. Ölblumen und ölsammelnde Bienen. Trop. Subtrop. Pflan-
zenwelt 7: 1–267.
VOGEL, S. 1988. Die Ölblumensymbiosen – Parallelismus und andere Aspekte
ihrer Entwicklung in Raum und Zeit. Z. Zool. Syst. Evol. 26: 341–362.
VOGEL, S. 1990. The role of scent glands in pollination. In Renner, S. S. (Ed.):
Smithsonian institution and the national science foundation. Washing-
ton, DC. pp. 60–180. Model Press (P) Ltd., New Delhi, India.
VOGEL, S., AND A. A. COCUCCI. 1995. Pollination of Basistemon (Scrophular-
iaceae) by oil-collecting bees in Argentina. Flora 190: 353–363.
VOGEL, S., AND I. C. MACHADO. 1991. Pollination of 4 sympatric species of An-
gelonia (Scrophulariaceae) by oil-collecting bees in NE Brazil. Plant Syst.
Evol. 178: 153–178.
Pollination Ecology of Mecardonia tenella 729
WANG, R. W., L. SHI, S. M. AI, AND Q. ZHENG. 2008. Trade-off between
reciprocal mutualists: Local resource availability-oriented interaction in
fig/fig wasp mutualism. Jour. Anim. Ecol. 77: 616–623.
WASER, N. M., L. CHITTKA, M. V. PRICE, N. M. WILLIAMS, AND J. OLLERTON.
1996. Generalization in pollination systems, and why it matters. Ecol-
ogy 77: 1043–1060.
WHITTEN, W. M., A. M. YOUNG, AND D. L. STERN. 1993. Nonfloral sources
of chemicals that attract male euglossine bees (Apidae, Euglossini).
J. Chem. Ecol. 19: 3017–3027.
WHITTEN, W. M., A. M. YOUNG, AND N. H. WILLIAMS. 1989. Function of glan-
dular secretions in fragrance collection by males euglossine bees (Apidae,
Euglossini). J. Chem. Ecol. 15: 1285–1295.
WILLIAMS, N. H., AND W. M. WHITTEN. 1983. Orchid floral fragrances and male
euglossine bees – methods and advances in the last sesquidecade. Bio-
logical Bulletin 164: 355–395.
WITTMANN, D., M. HOFFMANN, AND E. SCHOLZ. 1988. Southern distributional
limits of euglossine bees in Brazil linked to habitats of the Atlantic rain-
forest and subtropical rainforest (Hymenoptera, Apidae: Euglossini).
Entomol. Gen. 14: 53–60.
WOLFE, L. M., AND S. C. H. BARRETT. 1988. Temporal hanges in the pollinator
fauna of tristylous Pontederia-cordata, an aquatic plant. Can. J. Zol. 66:
1421–1424.