- Email: [email protected]
Deceptive pollination of Ionopsis utricularioides (Oncidiinae: Orchidaceae)
Accepted Manuscript Title: Deceptive pollination of Ionopsis utricularioides (Oncidiinae: Orchidaceae) Authors: Jo˜ao Marcelo Robazzi Bignelli Valente Aguiar, Emerson Ricardo Pansarin PII: DOI: Reference:
S0367-2530(18)30332-3 https://doi.org/10.1016/j.flora.2018.11.018 FLORA 51341
To appear in: Received date: Revised date: Accepted date:
25 May 2018 1 November 2018 21 November 2018
Please cite this article as: Aguiar JMRBV, Pansarin ER, Deceptive pollination of Ionopsis utricularioides (Oncidiinae: Orchidaceae), Flora (2018), https://doi.org/10.1016/j.flora.2018.11.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Deceptive pollination of Ionopsis utricularioides (Oncidiinae: Orchidaceae)
João Marcelo Robazzi Bignelli Valente Aguiar1*, Emerson Ricardo Pansarin2
IP T
João Marcelo Robazzi Bignelli Valente Aguiar1*
Present address: Programa de Pós-Graduação em Ecologia, Instituto de Biologia,
SC R
Universidade Estadual de Campinas, Cidade Universitária Zeferino Vaz - Barão
Programa de Pós-Graduação em Biologia Comparada, Departamento de Biologia,
N
1
U
Geraldo, 13083-865, Campinas, SP, Brazil.
A
Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São
2
M
Paulo, Av. Bandeirantes nº 3900, 14040-901, Ribeirão Preto, SP, Brazil. Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão
TE D
Preto, Universidade de São Paulo, Av. Bandeirantes nº 3900, 14040-901, Ribeirão Preto, SP, Brazil.
*
A
CC
EP
Corresponding author: [email protected]
Highlights
In Brazil, the flowers of Ionopsis utricularioides are nectarless.
Its flowers are pollinated by several bee species.
Brazilian populations of this orchid are self-incompatible.
1
The orchid flowers have similar colours to many other co-flowering species.
These results are consistent with non-specific deceptive pollination.
Abstract Oncidiinae is one of the largest and most diverse subtribes within Orchidaceae, and
IP T
many species of this group are known for relying on deceptive pollination through
Batesian floral mimicry. Ionopsis utricularioides is a widespread Oncidiinae orchid
SC R
that occurs from tropical South America to Florida, USA. The pollinators of this
species are still unknown, but a previous study in Puerto Rico showed that the species
U
is rewardless and self-compatible. Here we investigate the pollination biology of I.
N
utricularioides in Brazil. To determine if this species is a Batesian mimic we
A
specifically take into account the flower colour of this species and the co-flowering
M
plants in the community. The breeding system experiments revealed that Brazilian populations of I. utricularioides are completely self-incompatible and produces less
TE D
fruits than those from Puerto Rico under natural conditions. The flowers of I. utricularioides are also nectarless in Brazil and are pollinated by several bee species. The pollinarium is attached on the dorsal portion of the proboscis, while the
EP
pollinators put their head inside the flowers searching for nectar. Given that the flower
CC
colour is common to several other co-flowering species of the community, our results suggest that this orchid does not represent a case of Batesian mimicry, once it does
A
not mimic a specific model.
Key words: deceptive pollination, Epidendroideae, flower colour, food deception, Meliponini
2
1. Introduction About one third of orchid species are estimated to be pollinated by deceit (Ackerman, 1986). Deception occurs when the flowers do not provide any reward to the pollinators (Ackerman et al., 2011; Renner, 2006). Many deceptive mechanisms can be found in Orchidaceae, including sexual deception or oviposition-site mimicry,
IP T
but the most common mechanism is generalized food deception (Jersáková et al.
2006). In this case species are not specific mimics of other flowers, but rather present
SC R
general floral signals that attract generalist pollinators by their innate response to
these floral traits (Caballero-Villalobos et al., 2017; Johnson and Schiestl, 2016).
U
When deceptive species resemble particular rewarding flowers, and animals cannot
N
discriminate between models and mimics, the mechanism involved is referred to as
A
Batesian mimicry (Johnson and Schiestl, 2016). In Batesian mimicry, mimics should
M
resemble models beyond what it is expected from similarity among members of the same floral syndromes (Johnson and Schiestl, 2016). Therefore, the floral traits of the
TE D
deceptive species should imitate those of a rewarding co-occurring species and there should be overlap of specific pollinators between the rewarding and the rewardless species (Johnson and Schiestl, 2016; Pansarin et al., 2008; Vale et al., 2011).
EP
However, deceptive species could also exploit the interaction between pollinators and
CC
guilds of plants. In these cases the orchids do not mimic one specific plant species, but present floral traits that resemble those of a guild of multiple rewarding species
A
sharing the same pollinators (Jersáková et al., 2016; Johnson and Schiestl, 2016). Oncidiinae is one of the largest subtribes within Orchidaceae, presenting more
than 1,000 species within 70 genera (Chase, 2009). Most Oncidiinae orchids are known to be pollinated by deceit (Chase et al., 2009) and it is predicted that many species in this subtribe are Batesian mimics, especially in Gomesa spp., where
3
yellow-flowered orchids resemble the flowers of oil producing Malpighiaceae species (Papadopulos et al., 2013). However, evidence for the specific mechanism of pollinator attraction is currently lacking, especially due to the absence of confirmation of pollinator sharing between the Gomesa spp. and the Malpighiaceae species, a basic criteria for confirming Batesian mimicry (Johnson and Schiestl, 2016). Therefore,
IP T
more pollinator observations are needed to confirm shared pollinators in this system
(Papadopulos et al., 2013). Furthermore, Oncidiinae species are not always pollinated
SC R
trough deception. Most of the rewarding species of the subtribe present lipoidal
substances as a resource (Pansarin et al., 2017; Pansarin and Pansarin, 2011), whereas
U
other species produce floral nectar (Ackerman et al., 1994; Pansarin et al., 2015), or
N
fragrances that are collected by Euglossine bees (Singer et al., 2003).
A
Ionopsis utricularioides (Sw.) Lindl. is a widespread Oncidiinae species,
M
occurring from Florida to tropical South America (Ackerman, 1995). Although Ionopsis Kunth is a widespread genus, little is known about its pollination and
TE D
reproductive biology. According to Roubik (2000), Trigona fulventris (Meliponini) was collected with pollinaria of a species of Ionopsis, but it is unclear from which species or how it got attached to the bee. Montalvo and Ackerman (1987) show that I.
EP
utricularioides is pollinated by deceit in Puerto Rico, but pollinators are unknown.
CC
Also, in Puerto Rico, although the species is self-compatible, it forms few fruits under natural conditions, due to resources and pollinators limitations (Montalvo and
A
Ackerman, 1987). In this island, flowers of I. utricularioides vary from pale pink to pink or violet (Ackerman, 1995). We investigated the pollination system of Ionopsis utricularioides in Brazil, based on data on reproductive phenology, floral visitors, pollinator’s behaviour while visiting the flowers, presence of pollination resources and breeding system. We also
4
quantified flower colour, by measuring spectral reflectance of I. utricularioides flowers and compared it to the flower colours of several other species flowering at the same time as the orchid in the community. This serves as a measure for similarity between the deceptive orchid species and potential rewarding models, in order to investigate if I. utricularioides may present a Batesian mimic, as predicted for other
IP T
Oncidiinae species.
SC R
2. Materials and Methods 2.1 Study sites
U
Data was collected from plants maintained in cultivation at the Laboratório de
N
Biologia Molecular e Biossistemática de Plantas (LBMBP) Orchid House, Ribeirão
A
Preto, southeastern Brazil. Ionopsis utricularioides plants were collected in three
M
municipalities: São Simão (21° 28' 45'' S, 47° 33' 03'' W), Pradópolis (21° 21' 34'' S, 48° 03' 56'' W) and Dobrada (21° 31' 00'' S, 48° 23' 38'' W), state of São Paulo, Brazil.
TE D
Also, a natural population from the same region of the two other locations, Serra Azul city (21° 18' 39'' S, 47° 33' 56'' W), was used as an open pollination control for breeding experiments and for the floral reflectance measurements. The study areas
EP
present a climate characterized as “Cwa” (mesothermic with a dry winter season)
CC
according to Köppen (1948), and the vegetation is characterized by mesophytic
A
semideciduous forests (Pinto, 1989).
2.2 Flowering phenology The periods of inflorescence production, flower anthesis and fruit dehiscence were observed under natural conditions in the Serra Azul population during the 2012 flowering season. For the flower lifespan, 30 flowers (two plants; one inflorescence
5
per plant) were checked daily for anthesis in the greenhouse during the 2013 flowering season.
2.3 Pollinators and pollination mechanism During the 2012 flowering season, pollinator observations were carried at the
IP T
Serra Azul population. These were made during August 2 and 3, from 9:00 to 16:00 h, August 8 and 9, from 9:00 to 15:00 h, August 10, from 9:00 to 13:00 h, August 14 to
SC R
17, from 9:00 to 15:00 h and August 20, from 9:00 to 14:00, totalling 59h of
observation. During the 2013 flowering season, observations were carried out using
U
the cultivated plants at the garden of the LBMBP Orchid House, at the University of São Paulo, campus Ribeirão Preto, where I. utricularioides also naturally occurs (Pais
A
N
et al., 2000). These observations were made during July 30 to August 1st, from 8:00 to
M
16:00 h, August 2, from 9:00 to 12:00 h, August 6, from 8:00 to 16:00 h and August 8, from 13:00 to 16:00, totalling 38h of observation. Thus, 97 hours of observation
TE D
were accumulated during both flowering seasons. Floral visitors were photographed or collected using an entomological net. Pollinator details were photographed with a stereomicroscope Stereozoom Leica S8
EP
APO attached to a PC employing IM50 image analysis software. Caught insects were
CC
identified and stored at the “Camargo collection” (RPSP), University of São Paulo. After each flower visit, pollinarium removal or pollinia deposition on the stigma was
A
assessed. Five fresh flowers from five different plants of I. utricularioides were sectioned longitudinally in order to expose the spur lumen. The presence of nectar and secretion of exudates was checked using a stereomicroscope and afterwards the flowers were immersed in neutral red to evaluate if there was any tissue with metabolic activity in the spur (Dafni, 1992).
6
2.4 Spectral reflectance of flowers Spectral reflectance of Ionopsis utricularioides flowers (n = 7 individuals, one inflorescence per individual, three flowers per inflorescence) and flowers of all coflowering plants in the community (n = 3 individuals per species, one to three flowers
IP T
per individual) was measured at the Serra Azul site in 2016, using a USB4000
spectrophotometer (OceanOptics, Inc., Dunedin, FL, USA) calibrated between 300
SC R
and 700 nm, coupled with a deuterium–halogen light source (DH-2000; OceanOptics, Inc., Ostfildern, Germany). We performed measures of floral parts clearly visible for the pollinators: three measures of the labellum of each individual orchid and three
N
U
measures of the petals of each individual from the community. All of the reflectance measurements were taken at a 45° angle and at the same direction relative to the
M
A
flower structure, using barium sulphate as the white standard and a black chamber as the black standard. To determine how pollinators perceive floral colours, the
TE D
reflectance profiles were analysed with the colour hexagon model (Chittka, 1992), using the photoreceptor sensitivities of the stingless bee Melipona quadrifasciata (Menzel et al., 1989). We chose M. quadrifasciata as a model, since most of the floral
EP
visitors were Meliponini bees (see section 3.2 for further details). A standard daylight
CC
illumination and a standard function of green leaves as the background were used (Wyszecki and Stiles, 1982). Within this colour space model, distances between
A
points are indicative of an insect’s ability to discriminate between colours. Under natural field conditions, bees can reliably distinguish differences in colour of more than 0.1 hexagon units (Chittka et al., 1994; Chittka et al., 2001; Papadopulos et al., 2013). This value was used to determine if the flower colour of the orchid species was different from that of the flowers of the other species in the community. If they are
7
different or if there are multiple species with similar colours to Ionopsis flowers, Batesian mimicry might not be the deceptive pollination mechanism of this orchid (Jersáková et al., 2006; Johnson and Schiestl, 2016). We compared the distances between the individual orchids to evaluate floral colour polymorphism and also between the individual orchids and mean loci calculated for the other flowering
IP T
species. Since there were no differences greater than 0.1 hexagon units between
individuals of the same species from the other flowering species in the community,
SC R
we calculated a species mean measure in these cases. A mean measure for each orchid individual was calculated, but we did not calculate a species mean measure for the
U
orchid, once distances greater than 0.1 hexagon units were found (see section 3.3 for
N
further details). Hexagon model and colour loci distances calculations were performed
2.5 Breeding system and fruit set
M
A
with R 3.4.2 (R Core Team, 2017) using the pavo package (Maia et al., 2013).
TE D
The breeding system of I. utricularioides was investigated using 54 plants maintained in pots at the LBMBP Orchid House. Given the effect of resource limitation on fruit and seed set on cultivated plants found by Montalvo and Ackerman
EP
(1987) for this species, a maximum of only four flowers per plant were used for the
CC
breeding system experiments. Four kinds of controlled pollination treatments were performed: manual self-pollination (n = 30 flowers, 10 inflorescences, 10 plants),
A
autonomous self-pollination (n = 30 flowers, 10 inflorescences, 10 plants), crosspollination (n = 77 flowers, 24 inflorescences, 24 plants) and emasculation (n = 30 flowers, 10 inflorescences, 10 plants). For the autonomous self-pollination treatment, inflorescences were bagged with tulle fabric inside a netted greenhouse. Cross-
8
pollination treatments were performed between plants from different populations. Flowers were manipulated at the first day of anthesis. Fructification rates (number of fruits produced divided by the number of treated flowers) were quantified for all of the treatments performed and for control individuals from non-cultivated individuals at the Serra Azul population (n = 37
IP T
individuals). The results obtained were compared with the data available for the
Puerto Rico populations (Montalvo and Ackerman, 1987). From each fruit formed in
SC R
the manual treatments, as for the fruits formed under natural conditions, 200 seeds were analysed to quantify the percentage of potentially viable seeds, based on the
N
U
presence or absence of embryos (Pansarin and Pansarin, 2011).
A
3. Results
M
3.1 Flowering phenology
Ionopsis utricularioides started producing inflorescences in May and the first
TE D
flowers were open at the end of July. Each plant produced only one inflorescence. The flowering peak occurred in August and the flowering period ended at the beginning of October. Under natural conditions, each plant produced 3-194 flowers. Non-pollinated
EP
flowers lasted up to 25 days and fruit dehiscence occurred approximately 4 months
CC
after pollination.
A
3.2 Pollinators and pollination mechanism Many bee species were documented as flower visitors of I. utricularioides
(Table 1), however the observed pollinators were Paratetrapedia flaveola, Ceratina sp., Paratrigona lineata and Augochlora sp., since only those were able to remove and/or deposit pollinaria. In one individual of P. flaveola and in one of Ceratina sp.
9
more than one viscidium (two and three, respectively) attached to the proboscis was observed, indicating multiple successful visits (Fig. 1B, D). During the observations in 2012 at the Serra Azul population, only one visit of an unidentified Meliponini bee was recorded. Furthermore, another Meliponini bee, Nannotrigona testaceicornis, was captured with a pollinarium of Ionopsis
IP T
utricularioides attached to the proboscis. This bee was collected while gathering pollen from Hedyosmum brasiliense Miq. (Chloranthaceae), which is one of the
SC R
phorophytes for the studied orchid species in this studied area. During 2013, 27 bee
visits were recorded and at least eight pollinaria were removed during the visits. The
U
visits occurred during the day and were more frequent between 10h00 and 15h00. In
N
all visits the bee landed on the labellum and, following the nectar guides, reached for
A
the lip base, inserting the head into the flower, between the labellum and the column
M
(Fig. 1A, C). While trying to probe for nectar, the bees touched the viscidium with the dorsal portion of the proboscis removing the pollinarium.
TE D
During the floral reflectance measurements in 2016, three individuals of Scaptotrigona aff. depilis (Meliponini) were also observed visiting flowers of I. utricularioides in the Serra Azul population, but no pollinaria were removed.
EP
No secretion was recorded in the spur of I. utricularioides. Additionally, the
CC
neutral red test revealed no secretory tissue in the spur, indicating that the spur does
A
not produce nectar or any other resource (Fig. 1E-F).
3.3 Spectral reflectance To humans, flowers of Ionopsis utricularioides vary from whitish to purple colours. Flowers do not vary in colour within the same plant. In the hexagon model, the flowers are included in the bee-blue and bee-blue-green regions, and did not
10
present UV reflection. Most of the other flowers in the community are also included in the same region of the hexagon (Fig. 2A). Utricularia sp. and Asclepias curassavica L. had completely distinct flower colours from all of the I. utricularioides individuals (more than 0.1 hexagon units between colour loci), while Torenia thouarsii (Cham. & Schltdl.) Kuntze and Heliotropium sp. were the most similar in
IP T
colour to the orchid’s flowers (less than 0.1 hexagon units between colour loci) (Fig.
2B). Yet, most of the other species in the community also presented similar colours to
SC R
many of the I. utricularioides individuals sampled and cannot be visually
differentiated from different individuals of the orchid (Fig. 2B) (Table 2). Ionopsis
U
utricularioides also presents intraspecific colour polymorphism to the pollinators,
N
since flowers from different individuals of I. utricularioides can also present different
3.4 Breeding system and fruit set
M
A
colours, as shown by the Hexagon model (Fig. 2A-B).
TE D
In cross-pollinated flowers, fruit set was 55.84 ± 4.56% (mean ± S.E.). However, the emasculated, intact (bagged) and manually self-pollinated flowers formed no fruits, indicating that southeastern Brazilian populations of Ionopsis
EP
utricularioides are completely self-incompatible. Under natural conditions (open
CC
pollination), the fruit set by the end of the flowering season reached 5.35 ± 0.18%. Also, 48.65% of the plants analyzed formed no fruits in natural habitat (Table 3).
A
The percentage of potentially viable seeds yielded by the fruits obtained by the
cross-pollination treatment as trough natural fruit set had little variation and reached about 99% of potentially viable seeds in all cases.
4. Discussion
11
In this study we show that the rewardless flowers of Ionopsis utricularioides have similar colours to many of the co-flowering species of the studied community and are visited by many generalist bee species in Brazil. These features are consistent with generalized food deception or guild mimicry, but not with Batesian mimicry (Johnson and Schiestl, 2016). We also reveal that this species is self-incompatible in
IP T
Brazil, which is different from what was found in Puerto Rico by Montalvo and Ackerman (1987), where the plants are self-compatible.
SC R
Our results show that in the studied populations, I. utricularioides is pollinated by food deception, as pollinators exhibit a food foraging behaviour by searching for
U
nectar at the base of the lip, whereas the neutral red test showed that there is no nectar
N
secretion in the spur of this orchid species (Fig. 1E-F). Also, our results reveal that
A
Ionopsis utricularioides do not fit the premises for Batesian mimicry. Our colour
M
analysis revealed that the orchid species could be pollinated through generalized food deception or by a guild mimicry system. One of the most important premises for
TE D
generalized food deception is that the deceptive species advertises general floral signals that are typical for rewarding plant species (Jersáková et al., 2006). In a guild mimicry system, the orchids do not mimic one specific plant, but present floral traits
EP
that resemble those of several rewarding species in the community (Jersáková et al.,
CC
2016; Johnson and Schiestl, 2016). The bee-blue and bee-blue-green colour of the orchid was also the most common colour found in the other species of the community
A
(Fig. 2). In fact, this flower colour was also the most common in a study of 593 plant species, Chittka et al. (1994). Papadopulos et al. (2013) showed that this pattern of flower colour is also found in other communities with Oncidiinae species and suggest that these orchids would also be involved in a non-model deception.
12
We also showed that I. utricularioides has no specificity regarding its floral visitors. Pollinators include different species of solitary and social bees (Table 1), and flowering occurs for a long period. These features are consistent with a non-specific deceptive mechanism (Caballero-Villalobos et al., 2017; Jersáková et al., 2006). Many bees that visited the flowers of I. utricularioides are considered generalist
IP T
foragers in Brazil, such as Apis mellifera, Bombus sp., and the Meliponini bees
(Kleinert and Giannini, 2012) and seems likely that these animals visit both the
SC R
deceptive orchid and other plants in the community, which would be a further premise for the non-specific deceptive mechanism (Johnson and Schiestl, 2016). However, we
U
could not assess if pollinators were shared among community members, due to a
N
general scarcity of floral visitors in the community (cf. Papadopulos et al. 2013).
A
Thus, our results for I. utricularioides are consistent with a non-specific deceptive
M
pollination system, but more studies are needed to confirm if it is generalized food deception or if it is a case of guild mimicry. We specifically think that further field
TE D
observations, or the analysis of pollen identity on bees, are required to confirm pollinator sharing between rewarding plants and the orchids in this case. Also, investigating similarity in floral architecture and morphometry between the orchid and
EP
the co-flowering species could be useful to confirm a possible guild mimicry system.
CC
We found that I. utricularioides presents intraspecific colour polymorphism to the pollinators, which is a common feature of plants pollinated trough generalized
A
food deception (Ackerman et al., 2011; Jersáková et al., 2006). Many orchids pollinated through this mechanism present floral colour and/or floral fragrance polymorphism (Gigord et al., 2001; Juillet and Scopece, 2010; Salzmann et al., 2007). The underlying cause for intra-specific local variation could be due to selection
13
against pollinator learning behaviour to avoid flowers (Heinrich, 1975), but currently there is no strong evidence to support this hypothesis (Juillet and Scopece, 2010). When comparing the fruit set of Brazilian and Puerto Rican populations of Ionopsis utricularioides, we found an important difference (Table 3). Our study shows that in Brazil the plants are self-incompatible and the fruit set by the end of the
IP T
flowering season is 5.25% under natural conditions, with a high rate of potentially
viable seeds (99%), which is common for Oncidiinae (Pansarin and Pansarin, 2011;
SC R
Pansarin et al., 2018). In the Caribbean, the flowers are self-compatible and at the middle of the flowering period fruit set was 7.20% under natural conditions
U
(Montalvo and Ackerman, 1987). This latter study also argues that until the end of the
N
flowering period the fruit set could even double its value. Analyzing 447 plant
A
species, Sutherland (1986) found that self-compatible plants presented significantly
M
higher fruit set than self-incompatible species. In addition, although the low fruit set in orchids is often regarded to pollinator scarcity (Mickeliunas et al., 2006; Neiland
TE D
and Wilcock, 1998), this could also be a consequence of self-incompatibility (Cheng et al., 2009; Pansarin and Pansarin, 2011). Baker’s Law states that natural selection favours self-compatible populations
EP
in insular environments, when compared to continental locations (Baker, 1967, 1955).
CC
For autogamous species, the colonization of isolated environments is even more facilitated, once they do not depend on pollinator service. In Oececlades maculata
A
(Lindl.) Lindl., a widespread orchid that occurs both in continental and insular environments (Ackerman, 1995), a mixed mating system composed of crosspollination through butterflies and rain-assisted autogamy was found (Aguiar et al., 2012). This is probably related to its success in colonizing new and isolated environments. The argument of Busch (2011) about Baker’s Law that a dispersal
14
event associated with a bottleneck effect could favour the colonization of isolated environments by self-compatible plants, could explain what happened in the case of I. utricularioides. However, it is necessary to evaluate the self-compatibility of this species throughout its whole range of occurrence to confirm this hypothesis. We conclude that I. utricularioides is pollinated by food deception without
IP T
specific models, and presents differences in its self-compatibility, when comparing
Brazilian and Puerto Rican populations, which could have favoured the colonization
SC R
of the island by this species. Therefore, although it is predicted that many Onciidinae are Batesian mimics, our study shows a different situation. As this subtribe is
U
extremely diverse, we emphasize the importance of pollination biology studies in this
Acknowledgements
M
A
discriminating and learning floral features.
N
huge group of orchid species, especially considering the pollinator’s ability in
TE D
The authors acknowledges MSc. Nielson A.P. Salvador (USP) and MSc. Paulo R. de M. Cabral for helping with fieldwork, Dra. Silvia R. de M. Pedro and Dr. Sidnei Mateus (USP) for identifying the pollinators, Dr. Gustavo H. Shimizu and MSc.
EP
André V. Scatigna (UNICAMP) for identifying the plants and Dra. Marlies Sazima
CC
and MSc. Pedro J. Bergamo (UNICAMP) for providing assistance to the floral reflectance measures and analysis. JMRBVA was a Master student of the Programa
A
de Pós-Graduação em Biologia Comparada, FFCLRP, USP, during the development of this study.
Declarations of interest: none
15
Funding This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and by Fundação de Amparo à Pesquisa do Estado de São Paulo
A
CC
EP
TE D
M
A
N
U
SC R
IP T
(FAPESP; process number 2015/05919-8) [funding to JMRBVA].
16
References Ackerman, J.D. 1986. Systems in orchids. Lindleyana 1, 108-113. Ackerman, J.D. 1995. An orchid flora of Puerto Rico and the Virgin Islands, first ed. NYBG, New York.
variable than those offering a reward? Plant Syst. Evol. 293, 91–99.
SC R
https://doi.org/10.1007/s00606-011-0430-6.
IP T
Ackerman, J.D., Cuevas, A.A., Hof, D., 2011. Are deception-pollinated species more
Ackerman, J.D., Rodriguez-Robles, J.A., Melendez, E.J., 1994. A Meager Nectar
U
Offering by an Epiphytic Orchid is Better than Nothing. Biotropica 26, 44–49.
N
https://doi.org/10.2307/2389109.
A
Aguiar, J.M.R.B.V., Pansarin, L.M., Ackerman, J.D., Pansarin, E.R., 2012. Biotic
M
versus abiotic pollination in Oeceoclades maculata (Lindl.) Lindl. (Orchidaceae). Plant Species Biol. 27, 86–95. https://doi.org/10.1111/j.1442-
TE D
1984.2011.00330.x.
Baker, H.G., 1967. Support for Baker’s Law-As a Rule. Evolution (N. Y). 21, 853. https://doi.org/10.2307/2406780.
EP
Baker, H.G., 1955. Self-Compatibility and Establishment After “Long-Distance”
CC
Dispersal. Evolution (N. Y). 9, 347. https://doi.org/10.2307/2405656. Busch, J.W., 2011. Demography, pollination, and baker’s law. Evolution (N. Y). 65,
A
1511–1513. https://doi.org/10.1111/j.1558-5646.2011.01224.x.
Caballero-Villalobos, L., Silva-Arias, G.A., Buzatto, C.R., Nervo, M.H., Singer, R.B., 2017. Generalized food-deceptive pollination in four Cattleya (Orchidaceae: Laeliinae) species from Southern Brazil. Flora Morphol. Distrib. Funct. Ecol. Plants 234, 195–206. https://doi.org/10.1016/j.flora.2017.07.014.
17
Chase, M.W. 2009. Subtribe Oncidiinae, in: Pridgeon, A.M., Chase, M.W., Cribb, P.J., Rasmussen, F.N. (Eds.) Genera Orchidacearum, Vol. 5. Epidendroideae (part two). Oxford University Press, Oxford, pp. 211–394. Chase, M.W., Williams, N.H., De Faria, A.D., Neubig, K.M., Amaral, M.D.C.E.,
Orchidaceae): An expanded concept of Gomesa and a new genus
IP T
Whitten, W.M., 2009. Floral convergence in Oncidiinae (Cymbidieae;
Nohawilliamsia. Ann. Bot. 104, 387–402. https://doi.org/10.1093/aob/mcp067.
SC R
Cheng, J., Shi, J., Shangguan, F.Z., Dafni, A., Deng, Z.H., Luo, Y.B., 2009. The
pollination of a self-incompatible, food-mimic orchid, Coelogyne fimbriata
U
(Orchidaceae), by female Vespula wasps. Ann. Bot. 104, 565–571.
N
https://doi.org/10.1093/aob/mcp029.
A
Chittka, L., 1992. The colour hexagon: a chromaticity diagram based on
M
photoreceptor excitations as a generalized representation of colour opponency. J. Comp. Physiol. A 170, 533–543. https://doi.org/10.1007/BF00199331.
TE D
Chittka, L., Shmida, A., Troje, N., Menzel, R., 1994. Ultraviolet as a component of flower reflections, and the colour perception of hymenoptera. Vision Res. 34, 1489–1508. https://doi.org/10.1016/0042-6989(94)90151-1.
EP
Chittka, L., Spaethe, J., Schmidt, A., Hickelsberger, A. 2001. Adaptation, constraint,
CC
and chance in the evolution of flower color and pollinator color vision, in: Chittka, L., Thomson, J.D. (Eds.) Cognitive ecology of pollination. Cambridge
A
University Press, Cambridge, pp. 106 – 126.
Dafni, A. 1992. Pollination ecology: a practical approach, first ed. Oxford University Press, Oxford. Gigord, L.D., Macnair, M.R., Smithson, A. 2001. Negative frequency-dependent selection maintains a dramatic flower color polymorphism in the rewardless
18
orchid Dactylorhiza sambucina (L.) Soo. Proc. Natl Acad. Sci. U.S.A. 98, 62536255. Heinrich, B., 1975. Bee Flowers: A Hypothesis on Flower Variety and Blooming Times. Evolution (N. Y). 29, 325–334. https://doi.org/10.2307/2407220.
deceptive pollination in orchids. Biol. Rev. Camb. Philos. Soc. https://doi.org/10.1017/S1464793105006986.
IP T
Jersáková, J., Johnson, S.D., Kindlmann, P., 2006. Mechanisms and evolution of
SC R
Jersáková, J., Spaethe, J., Streinzer, M., Neumayer, J., Paulus, H., Dötterl, S.,
Johnson, S. D., 2016. Does Traunsteinera globosa (the globe orchid) dupe its
U
pollinators through generalized food deception or mimicry?. Bot. J. Linn. Soc.,
N
180, 269-294.
A
Johnson, S.D., Schiestl, F.P. 2016. Floral mimicry, first ed. Oxford University Press,
M
Oxford.
Juillet, N., Scopece, G., 2010. Does floral trait variability enhance reproductive
TE D
success in deceptive orchids? Perspect. Plant Ecol. Evol. Syst. https://doi.org/10.1016/j.ppees.2010.05.001. Kleinert, A.D.M.P., Giannini, T. C., 2012. Generalist bee species on Brazilian bee-
EP
plant interaction networks. Psyche 2012, 291519.
CC
https://doi.org/10.1155/2012/291519 Köppen, W. 1948. Climatologia, first ed. Ed. Fondo de Cultura e Economia, México.
A
Maia, R., Eliason, C.M., Bitton, P.P., Doucet, S.M., Shawkey, M.D., 2013. pavo: An R package for the analysis, visualization and organization of spectral data. Methods Ecol. Evol. 4, 906–913. https://doi.org/10.1111/2041-210X.12069. Menzel, R., Ventura, D.F., Werner, A., Joaquim, L.C.M., Backhaus, W., 1989. Spectral sensitivity of single photoreceptors and color vision in the stingless bee,
19
Melipona quadrifasciata. J. Comp. Physiol. A 166, 151–164. https://doi.org/10.1007/BF00193460. Mickeliunas, L., Pansarin, E.R., Sazima, M., 2006. Biologia floral, melitofilia e influência de besouros Curculionidae no sucesso reprodutivo de Grobya
258. https://doi.org/10.1590/S0100-84042006000200006.
IP T
amherstiae Lindl. (Orchidaceae: Cyrtopodiinae). Rev. Bras. Botânica 29, 251–
utricularioides (Orchidaceae). Biotropica 19, 24–31.
SC R
Montalvo, A.M., Ackerman, J.D., 1987. Limitations to fruit production in Ionopsis
Neiland, M.R.M., Wilcock, C.C., 1998. Fruit set, nectar reward, and rarity in the
U
Orchidaceae. Am. J. Bot. 85, 1657–1671. https://doi.org/10.2307/2446499.
N
Pais, M.P., Manso, A.D.G., Varanda, E.M. 2000. Uma flora ilustrada: guia para as
A
plantas do Museu do Café. Holos, Ribeirão Preto.
M
Pansarin, E.R., Alves-dos-Santos, I., Pansarin, L.M., 2017. Comparative reproductive biology and pollinator specificity among sympatric Gomesa (Orchidaceae:
TE D
Oncidiinae). Plant Biol. 19, 147–155. https://doi.org/10.1111/plb.12525. Pansarin, E. R., Bergamo, P. J., Ferraz, L. J., Pedro, S. R., Ferreira, A. W., 2018. Comparative reproductive biology reveals two distinct pollination strategies in
EP
Neotropical twig-epiphyte orchids. Plant Syst. Evol, 304, 793-806.
CC
https://doi.org/10.1007/s00606-018-1510-7 Pansarin, E.R., Pansarin, L.M., 2011. Reproductive biology of Trichocentrum
A
pumilum: An orchid pollinated by oil-collecting bees. Plant Biol. 13, 576–581. https://doi.org/10.1111/j.1438-8677.2010.00420.x.
Pansarin, E.R., Pansarin, L.M., Alves-dos-Santos, I., 2015. Floral features, pollination biology, and breeding system of Comparettia coccinea (Orchidaceae: Oncidiinae). Flora Morphol. Distrib. Funct. Ecol. Plants 217, 57–63.
20
https://doi.org/10.1016/j.flora.2015.09.008. Pansarin, L.M., Pansarin, E.R., Sazima, M., 2008. Reproductive biology of Cyrtopodium polyphyllum (Orchidaceae): A Cyrtopodiinae pollinated by deceit. Plant Biol. 10, 650–659. https://doi.org/10.1111/j.1438-8677.2008.00060.x. Papadopulos, A.S.T., Powell, M.P., Pupulin, F., Warner, J., Hawkins, J.A., Salamin,
IP T
N., Chittka, L., Williams, N.H., Whitten, W.M., Loader, D., Valente, L.M., Chase, M.W., Savolainen, V., 2013. Convergent evolution of floral signals
SC R
underlies the success of Neotropical orchids. Proc. R. Soc. B Biol. Sci. 280, 20130960–20130960. https://doi.org/10.1098/rspb.2013.0960.
U
Pinto, M.M. 1989. Levantamento fitossociológico de uma mata residual: Campus de
N
Jaboticabal da UNESP. Universidade Estadual Paulista, Jaboticabal.
A
Renner, S.S. 2006. Rewardless flowers in the angiosperms and the role of insect
M
cognition in their evolution, in: Waser, N.M., Orleton, J. (Eds.), Plant-Pollinator Interactions From Specialization to Generalization, The University of Chicago
TE D
Press, Chicago, pp 123-144.
Roubik, D.W., 2000. Deceptive orchids with Meliponini as pollinators. Plant Syst. Evol. 222, 271–279. https://doi.org/10.1007/BF00984106.
EP
Salzmann, C.C., Nardella, A.M., Cozzolino, S., Schiestl, F.P., 2007. Variability in
CC
floral scent in rewarding and deceptive orchids: The signature of pollinatorimposed selection? Ann. Bot. 100, 757–765.
A
https://doi.org/10.1093/aob/mcm161.
Singer, R.B., Koehler, S., Singer, R.B., Koehler, @bullet S, 2003. Notes on the pollination biology of Notylia nemorosa (Orchidaceae): do pollinators necessarily promote cross pollination? J Plant Res 116, 19–25. https://doi.org/10.1007/s10265-002-0064-4.
21
Sutherland, S., 1986. Patterns of Fruit-Set: What Controls Fruit-Flower Ratios in Plants? Evolution (N. Y). 40, 117–128. https://doi.org/10.1038/157619d0. Vale, Á., Navarro, L., Rojas, D., Álvarez, J.C., 2011. Breeding system and pollination by mimicry of the orchid Tolumnia guibertiana in Western Cuba. Plant Species Biol. 26, 163–173. https://doi.org/10.1111/j.1442-1984.2011.00322.x.
IP T
Wyszecki, G., Stiles, W.S. 1982. Color Science: Concepts and Methods, Quantitative
A
CC
EP
TE D
M
A
N
U
SC R
Data and Formulae, first ed. Wiley, NewYork.
22
IP T SC R U N A M TE D EP
Figure 1. Flowers and pollinators of Ionopsis utricularioides (Sw.) Lindl. (A)
CC
Ceratina sp. visiting the flower. Note how the insect inserts its head into the flower (arrow). (B) Ceratina sp. bearing two pollinaria (arrow heads) at the dorsal portion of the proboscis. (C) Paratetrapedia lineata visiting a flower. (D) Paratetrapedia lineata
A
carrying the pollinaria (arrow). In the detail, it is possible to see three viscidia (arrow heads) indicating visits to multiple flowers. (E) Flower of I. utricularioides longitudinal section stained with neutral red. (F) Detail of the spur stained with neutral red showing that the epidermis of the lumen does not present an evident metabolic activity (arrow). Scale bars: A, 5 mm; B, D, E, 2 mm; C, 10mm; F, 1 mm.
23
24
EP
CC
A TE D
IP T
SC R
U
N
A
M
op Io si s no 1 p Io si s no 2 p Io si s no 3 p Io si s 4 no p Io si s no 5 p Io si s no 6 ps is 7
A
Io n
B B
UV
Solanum sp.1 Solanum sp.2 Bacopa salzmannii Commelina sp. Echinodorus sp.
G
SC R
Heliotropium sp. Torenia thouarsii Cuphea sp. Utricularia sp.
IP T
Psidium guajava Asclepias curassavica
Acisanthera sp. Ionopsis 1 Ionopsis 2 Ionopsis 3 Ionopsis 4
U
Ionopsis 5
N
Ionopsis 6 Ionopsis 7
A
Figure 2. Colour analysis for Ionopsis utricularioides flowers and co-flowering E(G) species. (A) Bee colour hexagon model (Chittka 1992) using Melipona quadrifasciata
M
as a model. Triangles are I. utricularioides individuals and circles are the pooled data for other co-flowering species. Colours in the graph are a representation of human-
TE D
vision colours for the flowers. The inset shows the colour hexagon divided into sections that represent colour names as termed with respect to bee vision (B, blue; G, green; UV, ultraviolet). (B) Colour loci distances between the sampled species and I.
EP
utricularioides individuals (Ionopsis 1 to 7). Red dots indicate a distance higher than 0.1 hexagon units, indicating that bees can distinguish these colours. Blue dots
CC
indicate a distance lower than 0.1 hexagon units, meaning that both colours are similar for the bees. Note that even between individuals of I. utricularioides there are
A
differences in colour perception.
25
Table 1. Floral visitors and pollinators (pollinarium removal and/or deposition during the visit; marked with *) recorded on flowers of Ionopsis utricularioides. Species
Number of visits
Pollinator
1
-
Apidae
Apis mellifera scutellata
IP T
Apini
Bombus sp.
1
Ceratinini 6
U
Ceratina sp.
1
* -
N
Ceratinula sp.
-
SC R
Bombini
A
Meliponini
5
*
Nannotrigona testaceicornis
?1
*
Trigona spinipes
2
-
Scaptotrigona aff. depilis
3
-
6
*
5
*
TE D
M
Paratrigona lineata
EP
Tapinotaspidini
Paratetrapedia flaveola
CC
Halictidae
Augochlorini
A
Augochlora sp.
1
One individual of N. testaceicornis was captured with a pollinarium of I. utricularioides while foraging for pollen in another plant species. See Results for more information.
26
Table 2. Co-flowering species at the Serra Azul site and their colour for humans and
Bee-vision
N1
Psidium guajava L.
white
blue-green
9; 3
Asclepias curssavica L.
orange
UV-blue
9; 3
Solanum sp. 1
purple
blue
9; 3
Solanum sp. 2
purple
blue
6; 3
Bacopa salzmannii (Benth.) Wettst. ex Edwall
purple
blue
3; 3
Commelina sp.
purple
blue
3; 3
Echinodorus sp.
white
blue-green
9; 3
Heliotropium sp.
blue-green
9; 3
white
blue-green
4; 3
purple
blue
9; 3
yellow
UV
3; 3
purple
blue-green
6; 3
A M
Cuphea sp.
TE D
Utricularia sp.
Values are number of flowers; number of individuals.
A
CC
EP
1
N
white
Torenia thouarsii (Cham. & Schltdl.) Kuntze
Acisanthera sp.
SC R
Species
IP T
Human-vision
U
for bees as analysed by the colour Hexagon model.
27
Table 3. Fruit set results obtained from the experiments performed in southeastern Brazilian populations of Ionopsis utricularioides compared to the data presented for the Puerto Rico population. Values in parenthesis are fruit set/flowers. Treatments
Fruit Set (%) Puerto Rico1
55.84 (43/77)
89.70 (61/68)
Manual self-pollination
0 (0/30)
88.70 (55/62)
Spontaneous self-pollination
0 (0/30)
-
Emasculation
0 (0/30) 5.25 (72/1371)
7.20 (20/276)
Based on Montalvo and Ackerman (1987).
A
CC
EP
TE D
M
A
N
1
-
U
Open pollination
SC R
Cross-pollination
IP T
Southeastern Brazil
28
S0367-2530(18)30332-3 https://doi.org/10.1016/j.flora.2018.11.018 FLORA 51341
To appear in: Received date: Revised date: Accepted date:
25 May 2018 1 November 2018 21 November 2018
Please cite this article as: Aguiar JMRBV, Pansarin ER, Deceptive pollination of Ionopsis utricularioides (Oncidiinae: Orchidaceae), Flora (2018), https://doi.org/10.1016/j.flora.2018.11.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Deceptive pollination of Ionopsis utricularioides (Oncidiinae: Orchidaceae)
João Marcelo Robazzi Bignelli Valente Aguiar1*, Emerson Ricardo Pansarin2
IP T
João Marcelo Robazzi Bignelli Valente Aguiar1*
Present address: Programa de Pós-Graduação em Ecologia, Instituto de Biologia,
SC R
Universidade Estadual de Campinas, Cidade Universitária Zeferino Vaz - Barão
Programa de Pós-Graduação em Biologia Comparada, Departamento de Biologia,
N
1
U
Geraldo, 13083-865, Campinas, SP, Brazil.
A
Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São
2
M
Paulo, Av. Bandeirantes nº 3900, 14040-901, Ribeirão Preto, SP, Brazil. Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão
TE D
Preto, Universidade de São Paulo, Av. Bandeirantes nº 3900, 14040-901, Ribeirão Preto, SP, Brazil.
*
A
CC
EP
Corresponding author: [email protected]
Highlights
In Brazil, the flowers of Ionopsis utricularioides are nectarless.
Its flowers are pollinated by several bee species.
Brazilian populations of this orchid are self-incompatible.
1
The orchid flowers have similar colours to many other co-flowering species.
These results are consistent with non-specific deceptive pollination.
Abstract Oncidiinae is one of the largest and most diverse subtribes within Orchidaceae, and
IP T
many species of this group are known for relying on deceptive pollination through
Batesian floral mimicry. Ionopsis utricularioides is a widespread Oncidiinae orchid
SC R
that occurs from tropical South America to Florida, USA. The pollinators of this
species are still unknown, but a previous study in Puerto Rico showed that the species
U
is rewardless and self-compatible. Here we investigate the pollination biology of I.
N
utricularioides in Brazil. To determine if this species is a Batesian mimic we
A
specifically take into account the flower colour of this species and the co-flowering
M
plants in the community. The breeding system experiments revealed that Brazilian populations of I. utricularioides are completely self-incompatible and produces less
TE D
fruits than those from Puerto Rico under natural conditions. The flowers of I. utricularioides are also nectarless in Brazil and are pollinated by several bee species. The pollinarium is attached on the dorsal portion of the proboscis, while the
EP
pollinators put their head inside the flowers searching for nectar. Given that the flower
CC
colour is common to several other co-flowering species of the community, our results suggest that this orchid does not represent a case of Batesian mimicry, once it does
A
not mimic a specific model.
Key words: deceptive pollination, Epidendroideae, flower colour, food deception, Meliponini
2
1. Introduction About one third of orchid species are estimated to be pollinated by deceit (Ackerman, 1986). Deception occurs when the flowers do not provide any reward to the pollinators (Ackerman et al., 2011; Renner, 2006). Many deceptive mechanisms can be found in Orchidaceae, including sexual deception or oviposition-site mimicry,
IP T
but the most common mechanism is generalized food deception (Jersáková et al.
2006). In this case species are not specific mimics of other flowers, but rather present
SC R
general floral signals that attract generalist pollinators by their innate response to
these floral traits (Caballero-Villalobos et al., 2017; Johnson and Schiestl, 2016).
U
When deceptive species resemble particular rewarding flowers, and animals cannot
N
discriminate between models and mimics, the mechanism involved is referred to as
A
Batesian mimicry (Johnson and Schiestl, 2016). In Batesian mimicry, mimics should
M
resemble models beyond what it is expected from similarity among members of the same floral syndromes (Johnson and Schiestl, 2016). Therefore, the floral traits of the
TE D
deceptive species should imitate those of a rewarding co-occurring species and there should be overlap of specific pollinators between the rewarding and the rewardless species (Johnson and Schiestl, 2016; Pansarin et al., 2008; Vale et al., 2011).
EP
However, deceptive species could also exploit the interaction between pollinators and
CC
guilds of plants. In these cases the orchids do not mimic one specific plant species, but present floral traits that resemble those of a guild of multiple rewarding species
A
sharing the same pollinators (Jersáková et al., 2016; Johnson and Schiestl, 2016). Oncidiinae is one of the largest subtribes within Orchidaceae, presenting more
than 1,000 species within 70 genera (Chase, 2009). Most Oncidiinae orchids are known to be pollinated by deceit (Chase et al., 2009) and it is predicted that many species in this subtribe are Batesian mimics, especially in Gomesa spp., where
3
yellow-flowered orchids resemble the flowers of oil producing Malpighiaceae species (Papadopulos et al., 2013). However, evidence for the specific mechanism of pollinator attraction is currently lacking, especially due to the absence of confirmation of pollinator sharing between the Gomesa spp. and the Malpighiaceae species, a basic criteria for confirming Batesian mimicry (Johnson and Schiestl, 2016). Therefore,
IP T
more pollinator observations are needed to confirm shared pollinators in this system
(Papadopulos et al., 2013). Furthermore, Oncidiinae species are not always pollinated
SC R
trough deception. Most of the rewarding species of the subtribe present lipoidal
substances as a resource (Pansarin et al., 2017; Pansarin and Pansarin, 2011), whereas
U
other species produce floral nectar (Ackerman et al., 1994; Pansarin et al., 2015), or
N
fragrances that are collected by Euglossine bees (Singer et al., 2003).
A
Ionopsis utricularioides (Sw.) Lindl. is a widespread Oncidiinae species,
M
occurring from Florida to tropical South America (Ackerman, 1995). Although Ionopsis Kunth is a widespread genus, little is known about its pollination and
TE D
reproductive biology. According to Roubik (2000), Trigona fulventris (Meliponini) was collected with pollinaria of a species of Ionopsis, but it is unclear from which species or how it got attached to the bee. Montalvo and Ackerman (1987) show that I.
EP
utricularioides is pollinated by deceit in Puerto Rico, but pollinators are unknown.
CC
Also, in Puerto Rico, although the species is self-compatible, it forms few fruits under natural conditions, due to resources and pollinators limitations (Montalvo and
A
Ackerman, 1987). In this island, flowers of I. utricularioides vary from pale pink to pink or violet (Ackerman, 1995). We investigated the pollination system of Ionopsis utricularioides in Brazil, based on data on reproductive phenology, floral visitors, pollinator’s behaviour while visiting the flowers, presence of pollination resources and breeding system. We also
4
quantified flower colour, by measuring spectral reflectance of I. utricularioides flowers and compared it to the flower colours of several other species flowering at the same time as the orchid in the community. This serves as a measure for similarity between the deceptive orchid species and potential rewarding models, in order to investigate if I. utricularioides may present a Batesian mimic, as predicted for other
IP T
Oncidiinae species.
SC R
2. Materials and Methods 2.1 Study sites
U
Data was collected from plants maintained in cultivation at the Laboratório de
N
Biologia Molecular e Biossistemática de Plantas (LBMBP) Orchid House, Ribeirão
A
Preto, southeastern Brazil. Ionopsis utricularioides plants were collected in three
M
municipalities: São Simão (21° 28' 45'' S, 47° 33' 03'' W), Pradópolis (21° 21' 34'' S, 48° 03' 56'' W) and Dobrada (21° 31' 00'' S, 48° 23' 38'' W), state of São Paulo, Brazil.
TE D
Also, a natural population from the same region of the two other locations, Serra Azul city (21° 18' 39'' S, 47° 33' 56'' W), was used as an open pollination control for breeding experiments and for the floral reflectance measurements. The study areas
EP
present a climate characterized as “Cwa” (mesothermic with a dry winter season)
CC
according to Köppen (1948), and the vegetation is characterized by mesophytic
A
semideciduous forests (Pinto, 1989).
2.2 Flowering phenology The periods of inflorescence production, flower anthesis and fruit dehiscence were observed under natural conditions in the Serra Azul population during the 2012 flowering season. For the flower lifespan, 30 flowers (two plants; one inflorescence
5
per plant) were checked daily for anthesis in the greenhouse during the 2013 flowering season.
2.3 Pollinators and pollination mechanism During the 2012 flowering season, pollinator observations were carried at the
IP T
Serra Azul population. These were made during August 2 and 3, from 9:00 to 16:00 h, August 8 and 9, from 9:00 to 15:00 h, August 10, from 9:00 to 13:00 h, August 14 to
SC R
17, from 9:00 to 15:00 h and August 20, from 9:00 to 14:00, totalling 59h of
observation. During the 2013 flowering season, observations were carried out using
U
the cultivated plants at the garden of the LBMBP Orchid House, at the University of São Paulo, campus Ribeirão Preto, where I. utricularioides also naturally occurs (Pais
A
N
et al., 2000). These observations were made during July 30 to August 1st, from 8:00 to
M
16:00 h, August 2, from 9:00 to 12:00 h, August 6, from 8:00 to 16:00 h and August 8, from 13:00 to 16:00, totalling 38h of observation. Thus, 97 hours of observation
TE D
were accumulated during both flowering seasons. Floral visitors were photographed or collected using an entomological net. Pollinator details were photographed with a stereomicroscope Stereozoom Leica S8
EP
APO attached to a PC employing IM50 image analysis software. Caught insects were
CC
identified and stored at the “Camargo collection” (RPSP), University of São Paulo. After each flower visit, pollinarium removal or pollinia deposition on the stigma was
A
assessed. Five fresh flowers from five different plants of I. utricularioides were sectioned longitudinally in order to expose the spur lumen. The presence of nectar and secretion of exudates was checked using a stereomicroscope and afterwards the flowers were immersed in neutral red to evaluate if there was any tissue with metabolic activity in the spur (Dafni, 1992).
6
2.4 Spectral reflectance of flowers Spectral reflectance of Ionopsis utricularioides flowers (n = 7 individuals, one inflorescence per individual, three flowers per inflorescence) and flowers of all coflowering plants in the community (n = 3 individuals per species, one to three flowers
IP T
per individual) was measured at the Serra Azul site in 2016, using a USB4000
spectrophotometer (OceanOptics, Inc., Dunedin, FL, USA) calibrated between 300
SC R
and 700 nm, coupled with a deuterium–halogen light source (DH-2000; OceanOptics, Inc., Ostfildern, Germany). We performed measures of floral parts clearly visible for the pollinators: three measures of the labellum of each individual orchid and three
N
U
measures of the petals of each individual from the community. All of the reflectance measurements were taken at a 45° angle and at the same direction relative to the
M
A
flower structure, using barium sulphate as the white standard and a black chamber as the black standard. To determine how pollinators perceive floral colours, the
TE D
reflectance profiles were analysed with the colour hexagon model (Chittka, 1992), using the photoreceptor sensitivities of the stingless bee Melipona quadrifasciata (Menzel et al., 1989). We chose M. quadrifasciata as a model, since most of the floral
EP
visitors were Meliponini bees (see section 3.2 for further details). A standard daylight
CC
illumination and a standard function of green leaves as the background were used (Wyszecki and Stiles, 1982). Within this colour space model, distances between
A
points are indicative of an insect’s ability to discriminate between colours. Under natural field conditions, bees can reliably distinguish differences in colour of more than 0.1 hexagon units (Chittka et al., 1994; Chittka et al., 2001; Papadopulos et al., 2013). This value was used to determine if the flower colour of the orchid species was different from that of the flowers of the other species in the community. If they are
7
different or if there are multiple species with similar colours to Ionopsis flowers, Batesian mimicry might not be the deceptive pollination mechanism of this orchid (Jersáková et al., 2006; Johnson and Schiestl, 2016). We compared the distances between the individual orchids to evaluate floral colour polymorphism and also between the individual orchids and mean loci calculated for the other flowering
IP T
species. Since there were no differences greater than 0.1 hexagon units between
individuals of the same species from the other flowering species in the community,
SC R
we calculated a species mean measure in these cases. A mean measure for each orchid individual was calculated, but we did not calculate a species mean measure for the
U
orchid, once distances greater than 0.1 hexagon units were found (see section 3.3 for
N
further details). Hexagon model and colour loci distances calculations were performed
2.5 Breeding system and fruit set
M
A
with R 3.4.2 (R Core Team, 2017) using the pavo package (Maia et al., 2013).
TE D
The breeding system of I. utricularioides was investigated using 54 plants maintained in pots at the LBMBP Orchid House. Given the effect of resource limitation on fruit and seed set on cultivated plants found by Montalvo and Ackerman
EP
(1987) for this species, a maximum of only four flowers per plant were used for the
CC
breeding system experiments. Four kinds of controlled pollination treatments were performed: manual self-pollination (n = 30 flowers, 10 inflorescences, 10 plants),
A
autonomous self-pollination (n = 30 flowers, 10 inflorescences, 10 plants), crosspollination (n = 77 flowers, 24 inflorescences, 24 plants) and emasculation (n = 30 flowers, 10 inflorescences, 10 plants). For the autonomous self-pollination treatment, inflorescences were bagged with tulle fabric inside a netted greenhouse. Cross-
8
pollination treatments were performed between plants from different populations. Flowers were manipulated at the first day of anthesis. Fructification rates (number of fruits produced divided by the number of treated flowers) were quantified for all of the treatments performed and for control individuals from non-cultivated individuals at the Serra Azul population (n = 37
IP T
individuals). The results obtained were compared with the data available for the
Puerto Rico populations (Montalvo and Ackerman, 1987). From each fruit formed in
SC R
the manual treatments, as for the fruits formed under natural conditions, 200 seeds were analysed to quantify the percentage of potentially viable seeds, based on the
N
U
presence or absence of embryos (Pansarin and Pansarin, 2011).
A
3. Results
M
3.1 Flowering phenology
Ionopsis utricularioides started producing inflorescences in May and the first
TE D
flowers were open at the end of July. Each plant produced only one inflorescence. The flowering peak occurred in August and the flowering period ended at the beginning of October. Under natural conditions, each plant produced 3-194 flowers. Non-pollinated
EP
flowers lasted up to 25 days and fruit dehiscence occurred approximately 4 months
CC
after pollination.
A
3.2 Pollinators and pollination mechanism Many bee species were documented as flower visitors of I. utricularioides
(Table 1), however the observed pollinators were Paratetrapedia flaveola, Ceratina sp., Paratrigona lineata and Augochlora sp., since only those were able to remove and/or deposit pollinaria. In one individual of P. flaveola and in one of Ceratina sp.
9
more than one viscidium (two and three, respectively) attached to the proboscis was observed, indicating multiple successful visits (Fig. 1B, D). During the observations in 2012 at the Serra Azul population, only one visit of an unidentified Meliponini bee was recorded. Furthermore, another Meliponini bee, Nannotrigona testaceicornis, was captured with a pollinarium of Ionopsis
IP T
utricularioides attached to the proboscis. This bee was collected while gathering pollen from Hedyosmum brasiliense Miq. (Chloranthaceae), which is one of the
SC R
phorophytes for the studied orchid species in this studied area. During 2013, 27 bee
visits were recorded and at least eight pollinaria were removed during the visits. The
U
visits occurred during the day and were more frequent between 10h00 and 15h00. In
N
all visits the bee landed on the labellum and, following the nectar guides, reached for
A
the lip base, inserting the head into the flower, between the labellum and the column
M
(Fig. 1A, C). While trying to probe for nectar, the bees touched the viscidium with the dorsal portion of the proboscis removing the pollinarium.
TE D
During the floral reflectance measurements in 2016, three individuals of Scaptotrigona aff. depilis (Meliponini) were also observed visiting flowers of I. utricularioides in the Serra Azul population, but no pollinaria were removed.
EP
No secretion was recorded in the spur of I. utricularioides. Additionally, the
CC
neutral red test revealed no secretory tissue in the spur, indicating that the spur does
A
not produce nectar or any other resource (Fig. 1E-F).
3.3 Spectral reflectance To humans, flowers of Ionopsis utricularioides vary from whitish to purple colours. Flowers do not vary in colour within the same plant. In the hexagon model, the flowers are included in the bee-blue and bee-blue-green regions, and did not
10
present UV reflection. Most of the other flowers in the community are also included in the same region of the hexagon (Fig. 2A). Utricularia sp. and Asclepias curassavica L. had completely distinct flower colours from all of the I. utricularioides individuals (more than 0.1 hexagon units between colour loci), while Torenia thouarsii (Cham. & Schltdl.) Kuntze and Heliotropium sp. were the most similar in
IP T
colour to the orchid’s flowers (less than 0.1 hexagon units between colour loci) (Fig.
2B). Yet, most of the other species in the community also presented similar colours to
SC R
many of the I. utricularioides individuals sampled and cannot be visually
differentiated from different individuals of the orchid (Fig. 2B) (Table 2). Ionopsis
U
utricularioides also presents intraspecific colour polymorphism to the pollinators,
N
since flowers from different individuals of I. utricularioides can also present different
3.4 Breeding system and fruit set
M
A
colours, as shown by the Hexagon model (Fig. 2A-B).
TE D
In cross-pollinated flowers, fruit set was 55.84 ± 4.56% (mean ± S.E.). However, the emasculated, intact (bagged) and manually self-pollinated flowers formed no fruits, indicating that southeastern Brazilian populations of Ionopsis
EP
utricularioides are completely self-incompatible. Under natural conditions (open
CC
pollination), the fruit set by the end of the flowering season reached 5.35 ± 0.18%. Also, 48.65% of the plants analyzed formed no fruits in natural habitat (Table 3).
A
The percentage of potentially viable seeds yielded by the fruits obtained by the
cross-pollination treatment as trough natural fruit set had little variation and reached about 99% of potentially viable seeds in all cases.
4. Discussion
11
In this study we show that the rewardless flowers of Ionopsis utricularioides have similar colours to many of the co-flowering species of the studied community and are visited by many generalist bee species in Brazil. These features are consistent with generalized food deception or guild mimicry, but not with Batesian mimicry (Johnson and Schiestl, 2016). We also reveal that this species is self-incompatible in
IP T
Brazil, which is different from what was found in Puerto Rico by Montalvo and Ackerman (1987), where the plants are self-compatible.
SC R
Our results show that in the studied populations, I. utricularioides is pollinated by food deception, as pollinators exhibit a food foraging behaviour by searching for
U
nectar at the base of the lip, whereas the neutral red test showed that there is no nectar
N
secretion in the spur of this orchid species (Fig. 1E-F). Also, our results reveal that
A
Ionopsis utricularioides do not fit the premises for Batesian mimicry. Our colour
M
analysis revealed that the orchid species could be pollinated through generalized food deception or by a guild mimicry system. One of the most important premises for
TE D
generalized food deception is that the deceptive species advertises general floral signals that are typical for rewarding plant species (Jersáková et al., 2006). In a guild mimicry system, the orchids do not mimic one specific plant, but present floral traits
EP
that resemble those of several rewarding species in the community (Jersáková et al.,
CC
2016; Johnson and Schiestl, 2016). The bee-blue and bee-blue-green colour of the orchid was also the most common colour found in the other species of the community
A
(Fig. 2). In fact, this flower colour was also the most common in a study of 593 plant species, Chittka et al. (1994). Papadopulos et al. (2013) showed that this pattern of flower colour is also found in other communities with Oncidiinae species and suggest that these orchids would also be involved in a non-model deception.
12
We also showed that I. utricularioides has no specificity regarding its floral visitors. Pollinators include different species of solitary and social bees (Table 1), and flowering occurs for a long period. These features are consistent with a non-specific deceptive mechanism (Caballero-Villalobos et al., 2017; Jersáková et al., 2006). Many bees that visited the flowers of I. utricularioides are considered generalist
IP T
foragers in Brazil, such as Apis mellifera, Bombus sp., and the Meliponini bees
(Kleinert and Giannini, 2012) and seems likely that these animals visit both the
SC R
deceptive orchid and other plants in the community, which would be a further premise for the non-specific deceptive mechanism (Johnson and Schiestl, 2016). However, we
U
could not assess if pollinators were shared among community members, due to a
N
general scarcity of floral visitors in the community (cf. Papadopulos et al. 2013).
A
Thus, our results for I. utricularioides are consistent with a non-specific deceptive
M
pollination system, but more studies are needed to confirm if it is generalized food deception or if it is a case of guild mimicry. We specifically think that further field
TE D
observations, or the analysis of pollen identity on bees, are required to confirm pollinator sharing between rewarding plants and the orchids in this case. Also, investigating similarity in floral architecture and morphometry between the orchid and
EP
the co-flowering species could be useful to confirm a possible guild mimicry system.
CC
We found that I. utricularioides presents intraspecific colour polymorphism to the pollinators, which is a common feature of plants pollinated trough generalized
A
food deception (Ackerman et al., 2011; Jersáková et al., 2006). Many orchids pollinated through this mechanism present floral colour and/or floral fragrance polymorphism (Gigord et al., 2001; Juillet and Scopece, 2010; Salzmann et al., 2007). The underlying cause for intra-specific local variation could be due to selection
13
against pollinator learning behaviour to avoid flowers (Heinrich, 1975), but currently there is no strong evidence to support this hypothesis (Juillet and Scopece, 2010). When comparing the fruit set of Brazilian and Puerto Rican populations of Ionopsis utricularioides, we found an important difference (Table 3). Our study shows that in Brazil the plants are self-incompatible and the fruit set by the end of the
IP T
flowering season is 5.25% under natural conditions, with a high rate of potentially
viable seeds (99%), which is common for Oncidiinae (Pansarin and Pansarin, 2011;
SC R
Pansarin et al., 2018). In the Caribbean, the flowers are self-compatible and at the middle of the flowering period fruit set was 7.20% under natural conditions
U
(Montalvo and Ackerman, 1987). This latter study also argues that until the end of the
N
flowering period the fruit set could even double its value. Analyzing 447 plant
A
species, Sutherland (1986) found that self-compatible plants presented significantly
M
higher fruit set than self-incompatible species. In addition, although the low fruit set in orchids is often regarded to pollinator scarcity (Mickeliunas et al., 2006; Neiland
TE D
and Wilcock, 1998), this could also be a consequence of self-incompatibility (Cheng et al., 2009; Pansarin and Pansarin, 2011). Baker’s Law states that natural selection favours self-compatible populations
EP
in insular environments, when compared to continental locations (Baker, 1967, 1955).
CC
For autogamous species, the colonization of isolated environments is even more facilitated, once they do not depend on pollinator service. In Oececlades maculata
A
(Lindl.) Lindl., a widespread orchid that occurs both in continental and insular environments (Ackerman, 1995), a mixed mating system composed of crosspollination through butterflies and rain-assisted autogamy was found (Aguiar et al., 2012). This is probably related to its success in colonizing new and isolated environments. The argument of Busch (2011) about Baker’s Law that a dispersal
14
event associated with a bottleneck effect could favour the colonization of isolated environments by self-compatible plants, could explain what happened in the case of I. utricularioides. However, it is necessary to evaluate the self-compatibility of this species throughout its whole range of occurrence to confirm this hypothesis. We conclude that I. utricularioides is pollinated by food deception without
IP T
specific models, and presents differences in its self-compatibility, when comparing
Brazilian and Puerto Rican populations, which could have favoured the colonization
SC R
of the island by this species. Therefore, although it is predicted that many Onciidinae are Batesian mimics, our study shows a different situation. As this subtribe is
U
extremely diverse, we emphasize the importance of pollination biology studies in this
Acknowledgements
M
A
discriminating and learning floral features.
N
huge group of orchid species, especially considering the pollinator’s ability in
TE D
The authors acknowledges MSc. Nielson A.P. Salvador (USP) and MSc. Paulo R. de M. Cabral for helping with fieldwork, Dra. Silvia R. de M. Pedro and Dr. Sidnei Mateus (USP) for identifying the pollinators, Dr. Gustavo H. Shimizu and MSc.
EP
André V. Scatigna (UNICAMP) for identifying the plants and Dra. Marlies Sazima
CC
and MSc. Pedro J. Bergamo (UNICAMP) for providing assistance to the floral reflectance measures and analysis. JMRBVA was a Master student of the Programa
A
de Pós-Graduação em Biologia Comparada, FFCLRP, USP, during the development of this study.
Declarations of interest: none
15
Funding This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and by Fundação de Amparo à Pesquisa do Estado de São Paulo
A
CC
EP
TE D
M
A
N
U
SC R
IP T
(FAPESP; process number 2015/05919-8) [funding to JMRBVA].
16
References Ackerman, J.D. 1986. Systems in orchids. Lindleyana 1, 108-113. Ackerman, J.D. 1995. An orchid flora of Puerto Rico and the Virgin Islands, first ed. NYBG, New York.
variable than those offering a reward? Plant Syst. Evol. 293, 91–99.
SC R
https://doi.org/10.1007/s00606-011-0430-6.
IP T
Ackerman, J.D., Cuevas, A.A., Hof, D., 2011. Are deception-pollinated species more
Ackerman, J.D., Rodriguez-Robles, J.A., Melendez, E.J., 1994. A Meager Nectar
U
Offering by an Epiphytic Orchid is Better than Nothing. Biotropica 26, 44–49.
N
https://doi.org/10.2307/2389109.
A
Aguiar, J.M.R.B.V., Pansarin, L.M., Ackerman, J.D., Pansarin, E.R., 2012. Biotic
M
versus abiotic pollination in Oeceoclades maculata (Lindl.) Lindl. (Orchidaceae). Plant Species Biol. 27, 86–95. https://doi.org/10.1111/j.1442-
TE D
1984.2011.00330.x.
Baker, H.G., 1967. Support for Baker’s Law-As a Rule. Evolution (N. Y). 21, 853. https://doi.org/10.2307/2406780.
EP
Baker, H.G., 1955. Self-Compatibility and Establishment After “Long-Distance”
CC
Dispersal. Evolution (N. Y). 9, 347. https://doi.org/10.2307/2405656. Busch, J.W., 2011. Demography, pollination, and baker’s law. Evolution (N. Y). 65,
A
1511–1513. https://doi.org/10.1111/j.1558-5646.2011.01224.x.
Caballero-Villalobos, L., Silva-Arias, G.A., Buzatto, C.R., Nervo, M.H., Singer, R.B., 2017. Generalized food-deceptive pollination in four Cattleya (Orchidaceae: Laeliinae) species from Southern Brazil. Flora Morphol. Distrib. Funct. Ecol. Plants 234, 195–206. https://doi.org/10.1016/j.flora.2017.07.014.
17
Chase, M.W. 2009. Subtribe Oncidiinae, in: Pridgeon, A.M., Chase, M.W., Cribb, P.J., Rasmussen, F.N. (Eds.) Genera Orchidacearum, Vol. 5. Epidendroideae (part two). Oxford University Press, Oxford, pp. 211–394. Chase, M.W., Williams, N.H., De Faria, A.D., Neubig, K.M., Amaral, M.D.C.E.,
Orchidaceae): An expanded concept of Gomesa and a new genus
IP T
Whitten, W.M., 2009. Floral convergence in Oncidiinae (Cymbidieae;
Nohawilliamsia. Ann. Bot. 104, 387–402. https://doi.org/10.1093/aob/mcp067.
SC R
Cheng, J., Shi, J., Shangguan, F.Z., Dafni, A., Deng, Z.H., Luo, Y.B., 2009. The
pollination of a self-incompatible, food-mimic orchid, Coelogyne fimbriata
U
(Orchidaceae), by female Vespula wasps. Ann. Bot. 104, 565–571.
N
https://doi.org/10.1093/aob/mcp029.
A
Chittka, L., 1992. The colour hexagon: a chromaticity diagram based on
M
photoreceptor excitations as a generalized representation of colour opponency. J. Comp. Physiol. A 170, 533–543. https://doi.org/10.1007/BF00199331.
TE D
Chittka, L., Shmida, A., Troje, N., Menzel, R., 1994. Ultraviolet as a component of flower reflections, and the colour perception of hymenoptera. Vision Res. 34, 1489–1508. https://doi.org/10.1016/0042-6989(94)90151-1.
EP
Chittka, L., Spaethe, J., Schmidt, A., Hickelsberger, A. 2001. Adaptation, constraint,
CC
and chance in the evolution of flower color and pollinator color vision, in: Chittka, L., Thomson, J.D. (Eds.) Cognitive ecology of pollination. Cambridge
A
University Press, Cambridge, pp. 106 – 126.
Dafni, A. 1992. Pollination ecology: a practical approach, first ed. Oxford University Press, Oxford. Gigord, L.D., Macnair, M.R., Smithson, A. 2001. Negative frequency-dependent selection maintains a dramatic flower color polymorphism in the rewardless
18
orchid Dactylorhiza sambucina (L.) Soo. Proc. Natl Acad. Sci. U.S.A. 98, 62536255. Heinrich, B., 1975. Bee Flowers: A Hypothesis on Flower Variety and Blooming Times. Evolution (N. Y). 29, 325–334. https://doi.org/10.2307/2407220.
deceptive pollination in orchids. Biol. Rev. Camb. Philos. Soc. https://doi.org/10.1017/S1464793105006986.
IP T
Jersáková, J., Johnson, S.D., Kindlmann, P., 2006. Mechanisms and evolution of
SC R
Jersáková, J., Spaethe, J., Streinzer, M., Neumayer, J., Paulus, H., Dötterl, S.,
Johnson, S. D., 2016. Does Traunsteinera globosa (the globe orchid) dupe its
U
pollinators through generalized food deception or mimicry?. Bot. J. Linn. Soc.,
N
180, 269-294.
A
Johnson, S.D., Schiestl, F.P. 2016. Floral mimicry, first ed. Oxford University Press,
M
Oxford.
Juillet, N., Scopece, G., 2010. Does floral trait variability enhance reproductive
TE D
success in deceptive orchids? Perspect. Plant Ecol. Evol. Syst. https://doi.org/10.1016/j.ppees.2010.05.001. Kleinert, A.D.M.P., Giannini, T. C., 2012. Generalist bee species on Brazilian bee-
EP
plant interaction networks. Psyche 2012, 291519.
CC
https://doi.org/10.1155/2012/291519 Köppen, W. 1948. Climatologia, first ed. Ed. Fondo de Cultura e Economia, México.
A
Maia, R., Eliason, C.M., Bitton, P.P., Doucet, S.M., Shawkey, M.D., 2013. pavo: An R package for the analysis, visualization and organization of spectral data. Methods Ecol. Evol. 4, 906–913. https://doi.org/10.1111/2041-210X.12069. Menzel, R., Ventura, D.F., Werner, A., Joaquim, L.C.M., Backhaus, W., 1989. Spectral sensitivity of single photoreceptors and color vision in the stingless bee,
19
Melipona quadrifasciata. J. Comp. Physiol. A 166, 151–164. https://doi.org/10.1007/BF00193460. Mickeliunas, L., Pansarin, E.R., Sazima, M., 2006. Biologia floral, melitofilia e influência de besouros Curculionidae no sucesso reprodutivo de Grobya
258. https://doi.org/10.1590/S0100-84042006000200006.
IP T
amherstiae Lindl. (Orchidaceae: Cyrtopodiinae). Rev. Bras. Botânica 29, 251–
utricularioides (Orchidaceae). Biotropica 19, 24–31.
SC R
Montalvo, A.M., Ackerman, J.D., 1987. Limitations to fruit production in Ionopsis
Neiland, M.R.M., Wilcock, C.C., 1998. Fruit set, nectar reward, and rarity in the
U
Orchidaceae. Am. J. Bot. 85, 1657–1671. https://doi.org/10.2307/2446499.
N
Pais, M.P., Manso, A.D.G., Varanda, E.M. 2000. Uma flora ilustrada: guia para as
A
plantas do Museu do Café. Holos, Ribeirão Preto.
M
Pansarin, E.R., Alves-dos-Santos, I., Pansarin, L.M., 2017. Comparative reproductive biology and pollinator specificity among sympatric Gomesa (Orchidaceae:
TE D
Oncidiinae). Plant Biol. 19, 147–155. https://doi.org/10.1111/plb.12525. Pansarin, E. R., Bergamo, P. J., Ferraz, L. J., Pedro, S. R., Ferreira, A. W., 2018. Comparative reproductive biology reveals two distinct pollination strategies in
EP
Neotropical twig-epiphyte orchids. Plant Syst. Evol, 304, 793-806.
CC
https://doi.org/10.1007/s00606-018-1510-7 Pansarin, E.R., Pansarin, L.M., 2011. Reproductive biology of Trichocentrum
A
pumilum: An orchid pollinated by oil-collecting bees. Plant Biol. 13, 576–581. https://doi.org/10.1111/j.1438-8677.2010.00420.x.
Pansarin, E.R., Pansarin, L.M., Alves-dos-Santos, I., 2015. Floral features, pollination biology, and breeding system of Comparettia coccinea (Orchidaceae: Oncidiinae). Flora Morphol. Distrib. Funct. Ecol. Plants 217, 57–63.
20
https://doi.org/10.1016/j.flora.2015.09.008. Pansarin, L.M., Pansarin, E.R., Sazima, M., 2008. Reproductive biology of Cyrtopodium polyphyllum (Orchidaceae): A Cyrtopodiinae pollinated by deceit. Plant Biol. 10, 650–659. https://doi.org/10.1111/j.1438-8677.2008.00060.x. Papadopulos, A.S.T., Powell, M.P., Pupulin, F., Warner, J., Hawkins, J.A., Salamin,
IP T
N., Chittka, L., Williams, N.H., Whitten, W.M., Loader, D., Valente, L.M., Chase, M.W., Savolainen, V., 2013. Convergent evolution of floral signals
SC R
underlies the success of Neotropical orchids. Proc. R. Soc. B Biol. Sci. 280, 20130960–20130960. https://doi.org/10.1098/rspb.2013.0960.
U
Pinto, M.M. 1989. Levantamento fitossociológico de uma mata residual: Campus de
N
Jaboticabal da UNESP. Universidade Estadual Paulista, Jaboticabal.
A
Renner, S.S. 2006. Rewardless flowers in the angiosperms and the role of insect
M
cognition in their evolution, in: Waser, N.M., Orleton, J. (Eds.), Plant-Pollinator Interactions From Specialization to Generalization, The University of Chicago
TE D
Press, Chicago, pp 123-144.
Roubik, D.W., 2000. Deceptive orchids with Meliponini as pollinators. Plant Syst. Evol. 222, 271–279. https://doi.org/10.1007/BF00984106.
EP
Salzmann, C.C., Nardella, A.M., Cozzolino, S., Schiestl, F.P., 2007. Variability in
CC
floral scent in rewarding and deceptive orchids: The signature of pollinatorimposed selection? Ann. Bot. 100, 757–765.
A
https://doi.org/10.1093/aob/mcm161.
Singer, R.B., Koehler, S., Singer, R.B., Koehler, @bullet S, 2003. Notes on the pollination biology of Notylia nemorosa (Orchidaceae): do pollinators necessarily promote cross pollination? J Plant Res 116, 19–25. https://doi.org/10.1007/s10265-002-0064-4.
21
Sutherland, S., 1986. Patterns of Fruit-Set: What Controls Fruit-Flower Ratios in Plants? Evolution (N. Y). 40, 117–128. https://doi.org/10.1038/157619d0. Vale, Á., Navarro, L., Rojas, D., Álvarez, J.C., 2011. Breeding system and pollination by mimicry of the orchid Tolumnia guibertiana in Western Cuba. Plant Species Biol. 26, 163–173. https://doi.org/10.1111/j.1442-1984.2011.00322.x.
IP T
Wyszecki, G., Stiles, W.S. 1982. Color Science: Concepts and Methods, Quantitative
A
CC
EP
TE D
M
A
N
U
SC R
Data and Formulae, first ed. Wiley, NewYork.
22
IP T SC R U N A M TE D EP
Figure 1. Flowers and pollinators of Ionopsis utricularioides (Sw.) Lindl. (A)
CC
Ceratina sp. visiting the flower. Note how the insect inserts its head into the flower (arrow). (B) Ceratina sp. bearing two pollinaria (arrow heads) at the dorsal portion of the proboscis. (C) Paratetrapedia lineata visiting a flower. (D) Paratetrapedia lineata
A
carrying the pollinaria (arrow). In the detail, it is possible to see three viscidia (arrow heads) indicating visits to multiple flowers. (E) Flower of I. utricularioides longitudinal section stained with neutral red. (F) Detail of the spur stained with neutral red showing that the epidermis of the lumen does not present an evident metabolic activity (arrow). Scale bars: A, 5 mm; B, D, E, 2 mm; C, 10mm; F, 1 mm.
23
24
EP
CC
A TE D
IP T
SC R
U
N
A
M
op Io si s no 1 p Io si s no 2 p Io si s no 3 p Io si s 4 no p Io si s no 5 p Io si s no 6 ps is 7
A
Io n
B B
UV
Solanum sp.1 Solanum sp.2 Bacopa salzmannii Commelina sp. Echinodorus sp.
G
SC R
Heliotropium sp. Torenia thouarsii Cuphea sp. Utricularia sp.
IP T
Psidium guajava Asclepias curassavica
Acisanthera sp. Ionopsis 1 Ionopsis 2 Ionopsis 3 Ionopsis 4
U
Ionopsis 5
N
Ionopsis 6 Ionopsis 7
A
Figure 2. Colour analysis for Ionopsis utricularioides flowers and co-flowering E(G) species. (A) Bee colour hexagon model (Chittka 1992) using Melipona quadrifasciata
M
as a model. Triangles are I. utricularioides individuals and circles are the pooled data for other co-flowering species. Colours in the graph are a representation of human-
TE D
vision colours for the flowers. The inset shows the colour hexagon divided into sections that represent colour names as termed with respect to bee vision (B, blue; G, green; UV, ultraviolet). (B) Colour loci distances between the sampled species and I.
EP
utricularioides individuals (Ionopsis 1 to 7). Red dots indicate a distance higher than 0.1 hexagon units, indicating that bees can distinguish these colours. Blue dots
CC
indicate a distance lower than 0.1 hexagon units, meaning that both colours are similar for the bees. Note that even between individuals of I. utricularioides there are
A
differences in colour perception.
25
Table 1. Floral visitors and pollinators (pollinarium removal and/or deposition during the visit; marked with *) recorded on flowers of Ionopsis utricularioides. Species
Number of visits
Pollinator
1
-
Apidae
Apis mellifera scutellata
IP T
Apini
Bombus sp.
1
Ceratinini 6
U
Ceratina sp.
1
* -
N
Ceratinula sp.
-
SC R
Bombini
A
Meliponini
5
*
Nannotrigona testaceicornis
?1
*
Trigona spinipes
2
-
Scaptotrigona aff. depilis
3
-
6
*
5
*
TE D
M
Paratrigona lineata
EP
Tapinotaspidini
Paratetrapedia flaveola
CC
Halictidae
Augochlorini
A
Augochlora sp.
1
One individual of N. testaceicornis was captured with a pollinarium of I. utricularioides while foraging for pollen in another plant species. See Results for more information.
26
Table 2. Co-flowering species at the Serra Azul site and their colour for humans and
Bee-vision
N1
Psidium guajava L.
white
blue-green
9; 3
Asclepias curssavica L.
orange
UV-blue
9; 3
Solanum sp. 1
purple
blue
9; 3
Solanum sp. 2
purple
blue
6; 3
Bacopa salzmannii (Benth.) Wettst. ex Edwall
purple
blue
3; 3
Commelina sp.
purple
blue
3; 3
Echinodorus sp.
white
blue-green
9; 3
Heliotropium sp.
blue-green
9; 3
white
blue-green
4; 3
purple
blue
9; 3
yellow
UV
3; 3
purple
blue-green
6; 3
A M
Cuphea sp.
TE D
Utricularia sp.
Values are number of flowers; number of individuals.
A
CC
EP
1
N
white
Torenia thouarsii (Cham. & Schltdl.) Kuntze
Acisanthera sp.
SC R
Species
IP T
Human-vision
U
for bees as analysed by the colour Hexagon model.
27
Table 3. Fruit set results obtained from the experiments performed in southeastern Brazilian populations of Ionopsis utricularioides compared to the data presented for the Puerto Rico population. Values in parenthesis are fruit set/flowers. Treatments
Fruit Set (%) Puerto Rico1
55.84 (43/77)
89.70 (61/68)
Manual self-pollination
0 (0/30)
88.70 (55/62)
Spontaneous self-pollination
0 (0/30)
-
Emasculation
0 (0/30) 5.25 (72/1371)
7.20 (20/276)
Based on Montalvo and Ackerman (1987).
A
CC
EP
TE D
M
A
N
1
-
U
Open pollination
SC R
Cross-pollination
IP T
Southeastern Brazil
28