Biology of the Neotropical orchid genus Catasetum: A historical review on floral scent chemistry and pollinators

Accepted Manuscript Title: Biology of the Neotropical orchid genus Catasetum: a historical review on floral scent chemistry and pollinators Authors: Paulo Milet-Pinheiro, Gunter ¨ Gerlach PII: DOI: Reference:

S1433-8319(17)30106-3 http://dx.doi.org/doi:10.1016/j.ppees.2017.05.004 PPEES 25365

To appear in: Received date: Revised date: Accepted date:

27-4-2016 13-1-2017 29-5-2017

Please cite this article as: Milet-Pinheiro, Paulo, Gerlach, Gunter, ¨ Biology of the Neotropical orchid genus Catasetum: a historical review on floral scent chemistry and pollinators.Perspectives in Plant Ecology, Evolution and Systematics http://dx.doi.org/10.1016/j.ppees.2017.05.004 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.

Biology of the Neotropical orchid genus Catasetum: a historical review on floral scent chemistry and pollinators

Authors: Paulo Milet-Pinheiro a,1,*, Günter Gerlach b

a

Department of Fundamental Chemistry, Federal University of Pernambuco, Av. Prof.

Moraes Rego s/n, 50670-901, Recife, Brazil 1

Present Address: Department of Botany, Federal University of Pernambuco, Av. Prof.

Moraes Rego 1235, 50670-901 Recife, Brazil b

Botanischer Garten München-Nymphenburg, Menzinger Str. 61, 80638 München, Germany

* Corresponding author: [email protected]

Highlights 

Pollinators of more than 85% of all Catasetum species remain unknown



Flower scents of ca. 90% of all Catasetum species await chemical characterization



Evolution of floral scents is mediated by olfactory preferences of pollinators



Daily fluctuation in floral scents matches the foraging activity of pollinators



Possible sexual dimorphism in floral scents awaits experimental investigations

Abstract The Neotropical genus Catasetum is one of the most notable orchids because of its unusual reproductive strategy. In contrast to most orchids, all of the ca. 170 Catasetum species have sexually dimorphic, unisexual flowers involved in a highly specialized pollination mechanism. Flowers of Catasetum produce strong floral perfumes that act as both attractant and reward for male euglossine bees. While collecting perfumes, euglossine males may remove the pollinarium from a male flower and subsequently deposit it in the stigmatic slit of a female flower, resulting in pollination. Here we present an overview of the existing literature on floral scent chemistry and pollinators of Catasetum and add new reports on the pollinators of some species. We provide some insights into the ecology and evolution of floral scents in the genus Catasetum and suggest directions for future research. We hope this review will stimulate research not only on the ecology and evolution of Catasetum but also of the about 1000 species of fragrance-rewarding plants that are found in the Neotropics.

Keywords: Catasetinae; Eulaema; Euglossa; orchid bees; fragrance-rewarding plants; pollination.

Introduction The finding by Vogel (1966) that male euglossine bees do not gnaw on flowers (see, for example, Allen, 1950; Darwin, 1877; Ducke, 1902; Porsch, 1955), but rather collect and store floral scents in their hind tibiae for mating purposes, initiated the study of plants that provide floral volatiles as pollinator rewards. Between the 1970s and 1990s, numerous

publications began characterizing the chemistry of floral odors and proposed hypotheses of their ecological role and evolutionary origins (see, among others, Dodson 1970; Dodson et al., 1969; Gerlach and Schill, 1989; 1991; Hills et al., 1968; 1972; 1999; Kaiser, 1993; Lindquist et al., 1985; Sazima et al., 1993; Whitten and Williams, 1992; Whitten et al., 1986; 1988; Williams, 1983; Williams and Dodson, 1972; Williams and Whitten, 1999). These works focused on Orchidaceae, known for its high species diversity, but also included Araceae, Euphorbiaceae, Gesneriaceae, and Solanaceae. Among fragrance-rewarding orchids, the genus Catasetum (Orchidaceae) is one of the best studied groups of plants with respect to floral scent chemistry and pollination ecology. It has served as a model for understanding the role of floral scents in the evolution of fragrancerewarding plants as a whole (Hills et al., 1968; 1972; 1999; Lindquist et al., 1985; Whitten et al., 1986; 1988; Williams and Whitten, 1983). However, after the scientific frenzy that lasted until late 1990s, little information on basic aspects of the pollination ecology of Catasetum has been published (see supplementary material, Fig. S1 and Table S1). This is particularly curious because the number of species described since then has more than doubled, but to our knowledge studies investigating the pollination ecology of Catasetum in a classical approach have included only three species (Carvalho and Machado, 2002; Milet-Pinheiro et al., 2015; Murren, 2002). Half a century after the pioneering works that investigated the role of floral scents in the pollination ecology and evolution of Catasetum, no attempt has been made to review existing literature. In this article, we present an overview of all relevant information concerning floral scents and pollination ecology of Catasetum and add some new information on the pollinators of a few species. We also reevaluate the ecological and evolutionary significance of floral scents in the genus Catasetum using a multivariate statistical approach. Finally, we suggest future directions for research on Catasetum and other fragrance-rewarding flowers.

Floral biology and pollination ecology of Catasetum The Neotropical orchid genus Catasetum has long puzzled scientists, mainly because of its unusual sexual dimorphism (Darwin, 1877; Dodson and Frymire, 1961a). Different to most orchids, which have bisexual flowers, all Catasetum species bear unisexual flowers (van der Pijl and Dodson, 1969). Historically, Catasetum species have been considered dioecious, but this assumption was disproven by observation of plants in cultivation. Inflorescences of Catasetum bear usually male or female flowers (unisexual), but in very rare situations may form nonfunctional hermaphroditic flowers and/or flowers of both sexes. Sex expression is controlled by plant size and light intensity; large plants under strong light incidence normally develop female flowers, whereas younger and smaller plants, under moderate light incidence, develop male flowers (Dodson, 1962b; Gerlach, 2007; Gregg, 1975, 1982). Thus, Catasetum species may be better considered functionally dioecious. Female and male flowers of all Catasetum species are characterized by a pronounced sexual dimorphism (Fig. 1a, b) that is involved in a highly specialized pollination mechanism. The pollination mechanism of Catasetum aroused the curiosity of Darwin (Darwin, 1877), but it was only comprehensively described almost a century later (van der Pijl and Dodson, 1969). Flowers of Catasetum produce strong fragrances that are the only reward for pollinators. Male flowers generally bear an apparatus that forcibly ejects the pollinarium when a bee touches the sensitive antenna (projection of the rostellum) while trying to collect fragrances at the labellum. The pollinarium is expelled toward the pollinator with the viscidium striking the body of the bee (normally its thorax). The bee promptly flies away with an attached pollinarium; a subsequent visit to a receptive female flower, again for collecting fragrance, may result in pollination (for a scheme, see Dodson, 1962b) . Currently, our knowledge on the pollination mechanism of Catasetum species is well established. Following the pioneering works by Dodson and co-authors, further studies and

isolated field observations confirmed that the pollination mechanism, i.e., the catapult-like apparatus, works in the same way across the genus, even if the floral morphology varies considerably among species (Fulop, 2009; van der Pijl and Dodson, 1969). However, there are some subtle differences that are worth mentioning. Based on flower morphology, the genus Catasetum is divided into two subgenera: Pseudocatasetum, having rudimentary antennae (Fig. 1c), and Catasetum, having well-developed antennae (Fig. 1b, d). The subgenus Pseudocatasetum comprises only four species, which are all terrestrial with the exception of C. longifolium. The remaining species belong to the subgenus Catasetum and are mostly epiphytes (one exception: C. bergoldianum). With respect to the symmetry of the antennae, the subgenus Catasetum is divided into two sections. In section Isoceras, with more than 140 species, antennae are symmetric to each other (Fig. 1b). In the section Catasetum (formerly known as section Anisoceras; Mansfeld, 1932), with ca. 15-20 species, the antennae are asymmetrical and normally cross each other (Fig. 1d) (Romero et al., 2009; Romero and Jenny, 1993). Whereas pollinators of species of subgenus Catasetum receive the pollinaria on the dorsum of the body, in the subgenus Pseudocatasetum the pollinaria are glued to the bees ventrally. The force of the pollinarium striking the bee’s body is highest in section Catasetum, followed by section Isoceras and subgenus Pseudocatasetum (Gerlach pers. obs.), but exact measurements of force are still lacking.

In contrast to our established knowledge on the pollination mechanism of Catasetum, basic aspects of the pollination ecology of most species (e.g. identity of pollinators and floral scent chemistry) are poorly known. In Table 1, we provide a list of pollinators that have been reported or floral scent chemistry of species that have been investigated. In our literature survey, we found that the pollinators of only 26 Catasetum species are currently known at the species level, representing less than 15% of the described species for the genus (176 spp.;

Govaerts et al., 2015). The observation of Catasetum pollinators in situ has historically been hampered by many obstacles: 1) The genus Catasetum is composed almost exclusively of epiphytic species, which usually grow in trees at heights of 10-40 m ; 2) Catasetum is particularly diverse in Amazonian areas that are difficult to access; 3) Most species are rare (several are currently threatened) with low population densities; 4) Catasetum plants usually flower only once a year, and anthesis, at least of male flowers, lasts less than seven days; 5) Male flowers are required to identify plants to species; 6) Collection permits for Orchidaceae are difficult or impossible to obtain in many Latin American countries. While reviewing the literature, we were surprised at the astonishing amount of photographs of euglossine bees visiting flowers of Catasetum available on the internet. Surprisingly, many of the photographs are from Catasetum species with previously unknown pollinators. Although the identification of euglossine bees at the species level is not possible using photographs alone, we were able to determine the euglossine genera pollinating 17 Catasetum species (see Table 1 and Fig. 2). Based on our list, we found that pollination in Catasetum is performed predominantly by species of Euglossa (25 Catasetum spp.) and Eulaema (18 Catasetum spp.), whereas pollination by Eufriesea is reported for only two species (C. cernuum and C. fimbriatum). Representatives of these three bee genera differ morphologically and are easy to distinguish by the naked eye. In terms of body size, for example, the genus Euglossa (abbreviated here as Eg. to avoid confusion with the other euglossine genera) consists of more than 100 small to medium-sized species (8-18 mm long), Eufriesea (Ef.) of about 60 medium to large species (16-28 mm), and Eulaema (El.) of 20 large species (20 to 35 mm) (Ramirez et al., 2002). Given the highly specialized pollination mechanism of Catasetum, the size of pollinators is essential to assure that pollinarium transfer from male to female flowers occurs efficiently (Dodson, 1962b; 1978a). Thus, bees belonging to these genera might be seen as distinct pollinator guilds (see also Frankie et al., 1983). Indeed, within the genus Catasetum there is a clear partitioning in pollinating genera, at least

based on the literature information available so far. Species of the subgenus Pseudocatasetum and subgenus Catasetum section Anisoceras are all pollinated by Eulaema, whereas most but not all species of subgenus Catasetum section Isoceras are pollinated by Euglossa (Table 1; see also Romero et al., 2009). Exceptions include the sect. Isoceras species C. fimbriatum and C. cernuum, pollinated by Eufriesea, and C. planiceps, pollinated by Eulaema. Another interesting aspect discovered in our review is that Catasetum species pollinated by Eulaema are usually visited by two or more congeneric species but rarely by species of Euglossa. Similarly, Catasetum species pollinated by Euglossa are frequently visited by two or more congeneric species but rarely by Eulaema (Table 1; see also Whitten et al., 1986; 1988).

Floral scent chemistry in the genus Catasetum In terms of chemical characterization of floral scents, the obstacles mentioned above do not seem to be as limiting as they are for the survey of pollinators, because Catasetum species grow and flower very well in cultivation (Holst, 1999). Nevertheless, our review showed that the current knowledge on the chemical profile of floral scents of Catasetum is also limited. To date, 11 studies have investigated the floral scents of 30 species (ca. 17% of the known Catasetum species), of which those of only 23 species (12%) have been satisfactorily characterized (Table 2; see also Table S2 in supplementary material). Together, these studies reported 124 volatile compounds in the scent of Catasetum species, which belong to six compound classes, as categorized by Knudsen et al. (2006). Monoterpenes were the most diverse class with 44 compounds, followed by aromatics (38), sesquiterpenes (26), aliphatics (14), irregular terpenes (1) and N-bearing compounds (1). In terms of individual compounds, 1,8-cineole and α-pinene were the most commonly reported across species (16 species each), followed by β-pinene (12), (E)-dihydrocarvone (12), (E)-carvone epoxide (11), carvone (10) and p-cymene (10). Dihydrocarvone, carvone epoxide and carvone are responsible for the typical caraway scent of many Catasetum species.

The quality of chemical characterization of floral scent bouquets has increased considerably in the last years. In the seminal work by Hills et al. (1972), the floral scents of six species have had less than 25% of their total bouquet chemically characterized. In contrast, in more recent works (from the 1990s onward, with the advent of gas chromatography/mass spectrometry), all species have had at least 60% of the total scent bouquet characterized, most of them with more than 90% (Table S2). Clearly, the elucidation of new natural compounds, together with the continuous inclusion of mass spectra of compounds into commercial reference libraries, has greatly facilitated the chemical characterization of floral scent constituents in recent works. The intensive studies on the floral scent chemistry of Catasetum (and other perfume-rewarding orchids) might have already uncovered most of the volatile constituents of euglossine-pollinated plants. Nevertheless, even in recent works the number of compounds for which the identification has not been attributed is impressive (see, for example, Milet-Pinheiro et al., 2015). Most of these compounds are found in extremely low amounts, making their isolation and chemical elucidation challenging. The investigation of further Catasetum species will certainly bring to light some novelties. The complexity of floral scents in the species of Catasetum investigated so far varies considerably. When comparing species in which more than 95% of the scent profile has been characterized (see table 2), the number of compounds varied from one in C. micranthum (Hills et al., 1999) to 74 in C. uncatum (Milet-Pinheiro et al., 2015). This astonishing disparity in scent complexity across Catasetum species certainly reflects an inherent characteristic of each species; however, we cannot ignore that the sensitivity of analytical instrumentation has increased considerably in the last several years. Studies characterizing the floral scent chemistry of Catasetum were performed mainly between the 1970s and 2000s, when analytical instrument (gas chromatograph/mass spectrometers) were less sensitive than they are today. Although some species do have simple fragrances composed of one or two dominant compounds, the chemical complexity of other species is certainly underestimated.

The chemical profile of C. integerrimum, for example, was first investigated by Whitten et al. (1986) and more recently by Cancino and Damon (2007). In these studies, six and 28 volatile compounds were detected, respectively. Studies comparing the fragrance profiles of several Catasetum species and using the same analytical instrumentation might reveal the exact extent of chemical complexity in species of Catasetum. An exciting aspect of the floral fragrances of Catasetum (and other euglossine pollinated plants), which however remains completely neglected, is the chemistry of low-volatile compounds. To our best knowledge, floral scents of fragrance-rewarding plants have always been sampled using the headspace techniques, which is inappropriate to collect chemicals with high molecular weight and low vapor pressure (Pawliszyn, 2002; Tholl and Röse, 2006). In a recent study, Mittko et al. (2016) found that many of the dominant components of the leg pouches of euglossine males are semivolatiles (Kovats index > 1615). This suggests that fragrance-rewarding plants may produce low-volatile compounds to attract their euglossine pollinators. Fragrance-rewarding plants normally emit very strong floral scents, which are easily perceivable by the human olfaction (Gerlach and Schill, 1991; Hills et al., 1972; 1999; Williams and Whitten, 1983). However, there are also species that are practically scentless to the human olfaction and still extremely attractive to euglossine males. Some examples include representatives of the genera Catasetum (e.g. C. bicolor, C. boyi, C. callosum, C. cristatum, C. gladiatorium, C. lanciferum, C. schunkei), Paphinia spp. (except P. grandiflora), and most Sievekingia spp. (Milet-Pinheiro and Gerlach, unpubl. data). The investigation of low-volatile compounds (through solvent extracts of flowers, for example) represents a promising venue for future research on the chemical ecology of this specialized plant-pollinator mutualism.

Evolutionary and ecological aspects of floral scents in the genus Catasetum Floral scents as an isolating mechanism Reproductive isolation in Catasetum (and other fragrance-rewarding orchids) is achieved mainly by pollinator shifts, and floral scents are assumed to play a pivotal role in this sense (Gerlach and Schill, 1991; Hetherington-Rauth and Ramírez, 2016; Hills et al., 1972; Williams and Whitten, 1983). As common in angiosperms (Raguso, 2004b, 2008), floral scents of Catasetum tend to be species-specific, although dominated by a few major compounds, which are usually shared by several species. Most species have two or three major compounds that account together for more than 70% of the fragrance (see table 2). Individually, these compounds are normally potent attractants to many euglossine bees, but the attractiveness to individuals and species is extremely reduced as more compounds are added to mixtures, such that specific blends resembling the composition of selected plant species attract only a few pollinator species (Dodson et al., 1969; Hills et al., 1972; Williams and Dodson, 1972; Williams and Whitten, 1983). Accordingly, Catasetum species are known to attract only a few (normally congeneric) species of euglossine bees as pollinators in spite of the high euglossine diversity normally found in a given community, giving strong support to the hypothesis that floral scents act as primary barrier among otherwise interfertile species. The pivotal role of floral fragrances in pollinator shifts and as a reproductive isolating mechanism in Catasetum is incontestable. However, floral scents alone may not be enough to assure an effective reproductive isolation in Catasetum. Sympatric species usually produce similar fragrances, thereby attracting the same pollinator species (see, for example, Dodson, 1978a). In these cases, further isolating mechanisms (e.g. geographical, morphological/mechanical, temporal/seasonal), which may operate together, are necessary to prevent hybridization (Hills et al., 1972; Williams and Whitten, 1983).

Pollinator shift mediated by chemical changes has also been reported in other highly specialized pollination systems and is often assumed to act as a strong pre-pollination reproductive barrier (Johnson, 2006). Emblematic are the cases of the sexually deceptive orchid genera Ophrys (Ayasse et al., 2003; Schiestl et al., 1999) and Chiloglottis (Peakall et al., 2010; Schiestl et al., 2003), occurring in Europe and Australia, respectively. These orchids are normally pollinated by males of a single insect species (bees and wasps in Ophrys and wasps in Chiloglottis) and lure them by mimicking the sexual pheromone of conspecific females (Ayasse et al., 2003; Schiestl et al., 1999; 2003). In these studies, pollinator specificity has been found to have a strong chemical basis and chemical changes in floral scents has been shown to drive pollinator shift. Different to Catasetum, floral scents (a fraudulent sexual pheromone) seems to be enough to assure an effective isolation between sympatric species by attracting a single pollinator species, meaning that other isolating mechanisms are less relevant. Catasetum has a highly specialized pollination mechanism in which the size of pollinator is essential to assure the deposition of the pollinium into the stigmatic cavity of female flowers (Dodson, 1962b; Dodson and Frymire, 1961a; Hills et al., 1972). Because pollen is deposited as a package (pollinia), all microgametophytes of a flower might be lost if the pollinarium is removed by a floral visitor that does not fulfill the morphological requirements necessary for pollination (see for example Carvalho and Machado, 2002; Dodson, 1978a; Hills et al., 1972). Thus, attracting pollinators, while repelling other unwanted floral visitors, is essential in this specialized system. It seems plausible to hypothesize that the evolution of floral scents in Catasetum might be not only under the selective pressure of pollinators but also of other opportunistic non-pollinating euglossine males (see below) that could lead to pollinarium loss, resulting in reduced male fitness. The complexity of floral scents and the high diversity of euglossine species in a given community make it difficult to test this hypothesis. Investigation of Catasetum species occurring in Neotropical dry forests, where the diversity of euglossine

bees is much lower than in Neotropical rainforests (Lopes et al., 2007), would be an alternative to overcome these obstacles.

Evidence of pollinator-mediated selection in floral scents Recently, Ramírez et al. (2011) showed that fragrance-rewarding orchids originated at least three times independently after their fragrance-collecting bee mutualists, indicating that floral scents have evolved under the selective pressure of pre-existing sensory biases of their pollinators (see also Schiestl, 2010; Schiestl and Dötterl, 2012). Consequently, the evolution of floral scents in Catasetum might be strongly associated with the olfactory preferences of their euglossine pollinators. Given the well-defined partitioning of pollinating genera within Catasetum (see above), and the higher sensorial (i.e. olfactory receptors neurons) similarities between closely related euglossine species (Mitko et al., 2016), one might speculate that the evolution of floral fragrances within this genus (and likely in other fragrance-rewarding orchids) might have been shaped by olfactory adaptations with each euglossine genus. If this were true, we would expect that floral scents of species pollinated by Eulaema would resemble each other while differing considerably from those of species pollinated by Euglossa, and vice-versa. In order to test these assumptions, we performed a comparative and multivariate analyses based on the floral scent chemistry of the species included here. For this, we calculated first a Bray-Curtis similarity matrix (semi-quantitative approach, i.e. relative amount of each compound with respect to the total) and then performed a discriminant analysis (Canonical Analysis of Principal Coordinates, CAP) in the software Primer (v. 6.1.6; Clarke and Gorley, 2006). The analysis revealed interesting patterns with strong ecological and evolutionary implications. Floral fragrances of Catasetum group according to pollinating genera, i.e. Eulaema, Euglossa and Eufriesea (Fig. 3), and scent patterns differ significantly among groups [CAP permutation test: tr(Q_m'HQ_m) = 1.2, P <

0.001; delta_1^2 = 0.95, P < 0.001]. The leave-one-out allocation procedure, which provide a statistical estimate of the misclassification error between groups (Anderson et al., 2008), showed that 94% of the samples were assigned correctly to our pre-defined groups (i.e. pollinating genera) and this indicates that scent patterns predict well pollinating orchid bee genera. In order to depict better the pattern of scents of species pollinated by each bee genera, we performed a SIMPER and a PERMDISP analysis (again in Primer). We found that fragrances of species pollinated by Eulaema are less variable than those of species pollinated by Euglossa (SIMPER: average similarity 53% and 18%, respectively; PERMDISP: F1,14 = 22.9, P < 0.01), indicating a greater olfactory variability (i.e. chemical preference) in the genus Euglossa. Euglossine males are known to collect species-specific blends of compounds, which act as signal for recognition and/or fitness of conspecifics (Eltz et al., 1999; Pokorny et al., 2013; Zimmermann et al., 2009). In euglossine bees, chemical uniqueness is assumed to be difficult because of the elevated number of syntopic species (Eltz et al., 2008; Zimmermann et al., 2009) - a community can easily contain up to 50 species (Roubik and Hanson, 2004) - and this might be particularly true for Euglossa, which is by far the most diverse genus of the tribe with five times more species than Eulaema. Consequently, a greater olfactory variability in the genus Euglossa might be expected to assure reproductive isolation among species by means of chemical distinctness (Zimmermann et al., 2009) as would be expected for Eulaema, and this could explain why floral scents of species pollinated by Euglossa are more variable than those of species pollinated by Eulaema. Although natural selection might favor chemical distinctness between congeneric species (Weber et al., 2016; Zimmermann et al., 2009), we might still expect that species pollinated by Euglossa will have some typical scent patterns (although less evident than in Eulaema-pollinated species), mainly because males acquire fragrances from a limited number of scent sources available in nature. A recent study investigating hind tibia fragrances of males of 15 Euglossa species found that

chemical distinctness is accentuated between closely related species, whereas unrelated species usually share the same major compounds (homoplasy), indicating that chemical phenotypes in euglossine males do not evolve freely (Zimmermann et al., 2009). Information on the floral scent chemistry of species pollinated by Euglossa is scanty – among the 23 Catasetum species for which fragrances have been chemically characterized only 6 are pollinated by bees of this genus. Nevertheless, the SIMPER analysis revealed compounds that might be typical to species pollinated by both Euglossa and Eulaema. The remarkable dissimilarity (SIMPER: average dissimilarity 95%) between the fragrances of species pollinated by Euglossa and Eulaema can be explained mainly by six compounds, which together are responsible for about 75% of the total dissimilarity. Species pollinated by Euglossa are dominated by myrcene (on average 32% of the total scent discharge vs. 0% in species pollinated by Eulaema), 1,8-cineole (18% vs. 6%), and ipsdienol (15% vs. 0%), whereas those pollinated by Eulaema are dominated by (E)-carvone epoxide (39% vs. 0%), αpinene (19% vs. 2%), and benzyl acetate (6% vs. 0%). Assuming that the evolution of floral scents in Catasetum is mediated by chemical preferences of pollinators (Ramírez et al., 2011), we might expect that myrcene, 1,8-cineole and ipsdienol are only (or more) attractive to species of Euglossa, whereas (E)-carvone epoxide, α-pinene, and benzyl acetate are attractive to species of Eulaema. Biological assays in the field support this assumption only partially. The monoterpene myrcene, which was recorded in five of the six species pollinated by Euglossa but in none of the species pollinated by Eulaema, is reported to attract about 10 species of Euglossa and none of Eulaema (Table 3). Similarly, ipsdienol, a compound reported exclusively in species pollinated by Euglossa (three of six), attracts eight species of Euglossa but only one of Eulaema; only a single individual of El. nigrita has ever been collected using this compound (Whitten et al., 1988). In contrast to the two aforementioned compounds, 1,8-cineole, which was found in species

pollinated by both Euglossa (three of six species) and Eulaema (eight of ten species), is known to attract bees belonging to these two genera. However, even if attractive to seven species of Eulaema, 1,8-cineole seems to be a primary attractant of Euglossa bees because almost 80 species are known to collect this compound (Table 3). In the case of the compounds common to species pollinated by Eulaema, the scenario is somewhat different. (E)-carvone epoxide and benzyl acetate, for example, were recorded in 10 and 6 species of Catasetum pollinated by Eulaema, respectively, but in none of the species pollinated by Euglossa, indicating that these compounds selectively attract species of Eulaema. Curiously, these two compounds are not only attractive to Eulaema but also to Euglossa bees in behavioral tests (Table 3). Having in mind the well-defined pollinator partitioning observed for most Catasetum species, there might be some other constituent(s) in the scent profiles of species pollinated by Eulaema that repel Euglossa species and vice-versa (Whitten et al., 1986). α-Pinene, for instance, is known to reduce the attractiveness (in terms of number of individuals and species) of other highly attractive compounds when offered as a mixture (Dodson et al., 1969). This chiral compound is completely unattractive to euglossine bees as a racemate (Dodson et al., 1969; Williams and Whitten, 1983). However, the (-) isomer is a good attractant to Eulaema species, indicating that the (+) isomer acts as a repellent (Williams and Whitten, 1983). α-Pinene was found in much higher relative amounts in species pollinated by Eulaema (on average 19% in nine of ten species) than in species pollinated by Euglossa (on average 2% in four of six species), indicating that it could indeed play a differential role (i.e. attractant, repellent, deterrent) for bees of each genus. Unfortunately, the enantiomeric configuration of α-pinene in fragrance-rewarding plants is unknown, making any conclusion at this moment too speculative. Furthermore, for most compounds tested in the field so far, we only know whether they are attractive or not for a subset of euglossine species of a given community. Their possible negative (repellent, deterrent), neutral or combined (blends) effects on these bees remain poorly investigated.

Experimental studies integrating a detailed chemical characterization of floral scents in species pollinated by Eulaema and Euglossa, which includes the stereochemical configuration of chiral components and biological assays, ideally testing the behavioral meaning of compounds in a broader sense (attraction, repellence, deterrence), might help us to understand whether floral scents of Catasetum have also evolved under the selective pressure of opportunistic fragrance-collecting visitors. Myrcene, 1,8-cineole, benzyl acetate, and α-pinene are all compounds commonly reported as floral scent constituents in angiosperms (Knudsen et al., 2006). In contrast, (E)carvone epoxide and ipsdienol are reported exclusively in plants pollinated by euglossine males (Raguso, 2008; Whitten et al., 1986; 1988). Similar to what has been found in Catasetum, (E)-carvone epoxide and ipsdienol are dominant constituents of several other fragrance-rewarding plants pollinated by Eulaema and Euglossa, respectively. The occurrence of these compounds in plants belonging to different genera and even families indicates a case of convergent evolution mediated by chemical preference of pollinators (Whitten et al., 1986; 1988). According to Whitten et al. (1986), there is evidence of convergent evolution in floral scents even within the genus Catasetum, because species of subgenus Pseudocatasetum and subgenus Catasetum section Anisoceras emit similar fragrances attractive to species of Eulaema (Fig. 3). To avoid hybridisation here, the pollinaria in the subgenus Pseudocatasetum are attached ventrally on the pollinator’s body, whereas in subgenus Catasetum they are always attached dorsally. This reinforces the hypothesis that the evolution of floral scents in the genus Catasetum and other fragrace-rewarding flowers is mediated by chemical preference of euglossine pollinators. Unfortunately, the currently accepted classification of subgenera and sections in the genus Catasetum is still problematic and lacks molecular support, meaning that the hypothesis of pollinator-mediated selection of floral scents in Catasetum must be considered carefully.

Although the infrageneric delimitation of Catasetum into subgenera Pseudocatasetum and Catasetum is supported by phylogenetic studies (Pridgeon and Chase, 1998; Romero, 1990), the validity of the two sections within the subgenus Catasetum (i.e. Catasetum and Isoceras) is still controversial, as is the relationship among species (Fulop, 2009). Hopefully, a robust phylogeny of Catasetum at the species level will be possible in the near future. The recent advent of next-generation DNA sequencing technologies (NGS) (Mardis, 2008; Moore et al., 2006) has helped to increase the phylogenetic resolution in low taxonomic levels (i.e. genus, species, and populations) even of historically complicated orchid taxa (Harrison and Kidner, 2011; Parks et al., 2009; Yang et al., 2013). Therefore, future research using this modern technique might also shed some light on phylogenetic relationships within Catasetum.

Composition of floral scents in male and female flowers The remarkable sexual dimorphism in shape and color of Catasetum species, which once led renowned taxonomists to describe male and female individuals of a single species as different genera, has long aroused the curiosity of the scientific community (Darwin, 1877; Rolfe, 1890). However, its ecological and evolutionary meanings have remained poorly investigated until recently. In their seminal study, Romero and Nelson (1986) found indications that euglossine males avoided male flowers after forcible pollinarium emplacement (see section on pollination mechanism), but not to female flowers, and suggested that bees could discriminate flowers of different sexes by means of visual cues. According to these authors, "the aversion of the bee to pollinarium attachment and its avoidance of male flowers thereafter would promote pollination and apparently reflect competition among male flowers that probably evolved concurrently with sexual dimorphism". They also observed that the extent of morphological dimorphism among Catasetum species correlates with the degree of aversion caused by the male flowers (weight

of pollinarium). In other words, the heavier the pollinarium (in comparison to pollinator’s weight), the stronger the morphological sexual dimorphism. These results led authors to conclude that pollinarium emplacement on pollinator’s accounts for widespread morphological sexual dimorphism in Catasetum and for interspecific variations in its expression. The same evolutionary processes that are assumed to select for sexual dimorphism in shape and color are also likely to select for sexual divergence in floral scents of Catasetum. Thus, we might also expect sexual dimorphism in floral scents, if euglossine males learn to associate the "traumatic event" of pollinarium placement with a scent compound (or fragrance) that is typical for male flowers. Although this has never been tested experimentally, the learning ability of euglossine males (Eltz et al., 2005) indicates that this may be possible. In addition to the avoidance behavior of euglossine males, sexual dimorphism in Catasetum may be under the selective pressure of pollinator’s requirement for specific scent compounds. Euglossine males are known to change their scent preference after collecting a given compound intensively (Eltz et al., 2005). Thus, selection for sexual divergence might occur if 1) subtle differences in flower scent induces a greater flow of pollinators from male to female flowers (or vice versa) as males become ‘‘satiated’’ with a compound (or fragrance) that is typical for a given sex, and (2) this then results in increased plant fitness (see also Ashman, 2009). The direction of a pollinator from male to female is easy to explain. The euglossine male is shocked by the forcible placement of the pollinarium and is disturbed while collecting fragrances. Later on at the female flower, which does not have the pollinarium-triggering mechanism, the bee collects the floral scents without being disturbed. Thus, euglossine males starting scent collection in female flowers will never have the need to look for male ones if there isn’t any additional seduction.

Possible sexual dimorphism in floral scents of Catasetum, as well as its ecological and evolutionary meanings, has been investigated for only one species. In 2015, Milet-Pinheiro and collaborators investigated the pollination ecology of C. uncatum and found that frequency of visits by Euglossa pollinators to female flowers is much higher than to male flowers. Given the similar blooming period of male and female inflorescences and the higher number of male flowers in the population, the authors suggested that bees discriminate flowers of different sexes, preferring female flowers. However, they did not detect clear sexual dimorphism (either in the total amount or in the volatile blend), indicating that selection might act to reduce divergence between floral scents of male and female flowers (see also Ashman, 2009; Dötterl et al., 2014; Soler et al., 2012). In Catasetum (and fragrance-rewarding plants with unisexual flowers), the attraction of specific euglossine pollinators is known to be mediated by species-specific scent compositions (Hills et al., 1972), meaning that substantial changes in the scent of flowers of one of the sexes could disrupt the interaction with their specific pollinators and lead to reproductive failure in the plant. Thus, selection for sexual divergence might act with respect to other cues that are not directly involved in the primary attraction of euglossine pollinators; euglossine males might therefore rely on floral cues other than scents (e.g. color, shape) to discriminate male from female flowers (Milet-Pinheiro et al., 2015). Although plausible, this conclusion should be considered carefully and might not be extrapolated for Catasetum as a whole. Catasetum uncatum is a species with a moderate sexual dimorphism in color and shape, and scent dimorphism may be subtle accordingly. This is particularly critical because the number of scent samples collected from male (n=6) and female flowers (n=5) was relatively low, so sexual dimorphism in floral scents of C. uncatum may have been masked. Clearly, further studies investigating the floral scents of male and female flowers of several Catasetum species, preferentially presenting different degrees of morphological dimorphism, are still necessary to establish whether floral scent in Catasetum is a sexually dimorphic trait and, if so, to what extent.

Daily fluctuation in flower scent emission In Catasetum and other fragrance-rewarding orchids, it has long been assumed that emission of flower scents is higher during the morning, decreasing remarkably at the afternoon. However, the evolutionary meaning of this daily fluctuation remains elusive. Historically, daily fluctuation in scent emission in fragrance-rewarding plants has been based mainly on human olfaction (Carvalho and Machado, 2002; Dodson, 1978a; Hills, 1989; Hills and Williams, 1990; Janzen, 1981b; Martini et al., 2003; van der Pijl and Dodson, 1969), making conclusions about its evolutionary meaning too speculative. In 1999, Hills and Williams were the first to investigate the fragrance cycle of a fragrance-rewarding plant (Clowesia rosea: Orchidaceae) using chemical analytical methods for quantification. They found a higher emission of scent during the morning (with a peak at 10:00h), but did not venture further into the evolutionary significance of this fluctuation. In a more recent work, also using chemical analytical methods for quantification, Milet-Pinheiro et al. (2015) found that scent emission in flowers of C. uncatum is almost three times higher in the morning than in the afternoon and that the activity of pollinators in flowers (in this case two species of Euglossa) matches well with this daily fluctuation in scent emission. These authors concluded that the coordination of scent emission with periods of higher activity of pollinators would be a strategy to save the high energetic cost of scent production when the frequency of visits to flowers is expected to be low (Ashman and Schoen, 1994, 1996). As euglossine males do not specialize on a single fragrance source, whereas fragrance-rewarding plants usually depend on a few species as pollinators, it is much more likely that daily fluctuation in scent emission has evolved to match the male euglossine activity than vice-versa (Armbruster and McCormick, 1990). Although persuasive, this hypothesis awaits experimental support. Catasetum species emerge as an excellent model for testing this. In nature, foraging activity of bees is strongly associated

with body size and color; larger and darker bees normally initiate foraging earlier than their smaller and brighter counterparts do (Heinrich, 1993; Pereboom and Biesmeijer, 2003; Roubik, 1989). Accordingly, field observations in species of Catasetum pollinated by Eulaema indicate that activity of pollinators is higher between 06:00 and 09:00 (Carvalho and Machado, 2002; Dodson, 1978a; Janzen, 1981a), earlier than the peak of activity of pollinators in species pollinated by Euglossa (Milet-Pinheiro et al., 2015). Thus, if daily fluctuation were indeed a strategy to save energy when activity of pollinators is low, species pollinated by Eulaema would tend to emit more scent earlier in the morning than species pollinated by Euglossa. Experimental studies investigating the daily fluctuation in scent emission of Catasetum species pollinated by different euglossine genera, and simultaneously recording the frequency of pollinators, might shed light on this aspect. This mechanism could also increase reproductive isolation in species pollinated by bees of different genera. Effect of pollinarium removal/deposition in floral scent emission In angiosperms, the event of pollination usually triggers a series of physiological changes that affect many flower features, such as longevity, morphology, color, and scents (Gori, 1983). The changes in flower features following pollen deposition, known as post-pollination phenomena, normally result in a reduced attractiveness to pollinators (Doorn, 1997) and are assumed to be a strategy to save the high cost of floral maintenance when pollinators are no longer necessary (Ashman and Schoen, 1994, 1996). Floral scents play a pivotal role in attracting pollinators (Dötterl and Vereecken, 2010; 2004a; Raguso, 2004b, 2008), and their production obviously has some energetic cost, which could otherwise be allocated to other physiological needs of the plants (Ashman and Schoen, 1994, 1996). In the case of fragrancerewarding orchids, this cost might be much more accentuated because scents are not only the attractant but also the reward for pollinators (Vogel, 1990). Not surprisingly, reduction of scent emission following pollinarium removal/deposition, as well as other changes in flower

features, are repeatedly reported among fragrance-rewarding orchids (Dodson, 1965b; Dodson and Frymire, 1961a, b; Martini et al., 2003; van der Pijl and Dodson, 1969), including Catasetum species (Carvalho and Machado, 2002; Dodson, 1962b; 1978a; Janzen, 1981b; Milet-Pinheiro et al., 2015). In Catasetum, and other fragrance-rewarding orchids with unisexual flowers, both pollinarium removal and deposition in male and female flowers, respectively, initiate a cascade of physiological changes that includes the cessation of scent emission within a few hours. These observations were initially based on human olfaction (Carvalho and Machado, 2002; Dodson, 1962b; 1978a; Janzen, 1981b) but has recently received experimental support of chemical analytical methods (Milet-Pinheiro et al., 2015). In monecious orchid flowers, the effect of pollinarium removal might be different. In the genus Gongora, for instance, flowers are protandric, i.e. the pollinarium is first removed by the pollinator and only after that does the stigma become receptive (Dodson, 1962a; Dodson and Frymire, 1961a; HetheringtonRauth and Ramírez, 2015). Thus, pollinarium removal alone should not interfere in scent emission; otherwise flowers in female phase would be no longer attractive to pollinators, and pollination would not occur. Accordingly, in Gongora quinquenervis Ruíz & Pavón interruption of scent emission is reported to occur only after pollinarium deposition, a moment in which pollinators are not necessary anymore (Martini et al., 2003). It is probable that the species investigated in that study in NE-Brazil was G. vitorinoana Chiron & L.C. Menezes because G. quinquenervis does not occur there.

Concluding remarks and future perspectives Among fragrance-rewarding orchids, Catasetum is the most investigated, partly because of its high species diversity compared to other genera but also because of its bizarre and unusual reproductive strategies, which have long intrigued scientists. Nevertheless, many gaps in our

current knowledge remain. We encourage studies of Catasetum species and their pollinators (preferentially at the species level) through field observations, as well as about chemical profile of the floral scents of Catasetum species. The integration of this information with a well-supported phylogeny at the species level will provide the necessary background to understand how male euglossine pollinators have shaped the evolution of floral scents of Catasetum and to test hypotheses about macroevolution, diversification rates and mechanisms of speciation. This review might serve as a basis for future research not only on the ecology and evolution of Catasetum but also of fragrance-rewarding plants as a whole.

Acknowledgments We thank Alec Pridgeon and Mark W. Whitten for the critical reading of the manuscript and for linguistic advice, the orchid growers Juan Fernández Gómez, Valdir Sanches, Luiz Valter and Luiz Filipe Varella for gently providing photographs and information on the pollinators of some Catasetum species. This work was supported by the Fundação de Amparo à Pesquisa do Estado de Pernambuco (FACEPE), which provided a grant to P. Milet-Pinheiro (BFP-00282.03/10).

Literature cited Ackerman, J.D., 1983. Diversity and seasonality of male euglossine bees (Hymenoptera: Apidae) in Central Panamá. Ecology 64, 274-283. Ackerman, J.D., Roubik, D.W., 2012. Can extinction risk help explain plant–pollinator specificity among euglossine bee pollinated plants? Oikos 121, 1821-1827. Allen, P., 1950. Pollination in Coryanthes speciosa. Amer. Orchid Soc. Bull. 19, 528-536.

Anderson , M.J., Gorley, R.N., Clarke, K.R., 2008. PERMANOVA+ for PRIMER: Guide to software and statistical methods. PRIMER-E, Plymouth, UK. Armbruster, W.S., McCormick, K.D., 1990. Diel foraging patterns of male euglossine bees: ecological causes and evolutionary responses by plants. Biotropica 22, 160-171. Ashman, T.L., 2009. Sniffing out patterns of sexual dimorphism in floral scent. Funct. Ecol. 23, 852–862. Ashman, T.L., Schoen, D.J., 1994. How long should flowers live. Nature 371, 788-790. Ashman, T.L., Schoen, D.J., 1996. Floral longevity: fitness consequences and resource costs, in: Barrett, S.C.H., Lloyd, D.G. (Eds.), Floral Biology. Chapman and Hall, New York, pp. 112-139. Ayasse, M., Schiestl, F.P., Paulus, H.F., Ibarra, F., Francke, W., 2003. Pollinator attraction in a sexually deceptive orchid by means of unconventional chemicals. Proc. R. Soc. B 270, 517522. Cancino, A.D.M., Damon, A., 2007. Fragrance analysis of euglossine bee pollinated orchids from Soconusco, south-east Mexico. Plant Species Biol. 22, 129-134. Carvalho, R., Machado, I.C., 2002. Pollination of Catasetum macrocarpum (Orchidaceae) by Eulaema bombiformis (Euglossini). Lindleyana 17, 85-90. Clarke, K.R., Gorley, R.N., 2006. Primer v6: User Manual/Tutorial. Primer-E, Plymouth. Darwin, C., 1877. The various contrivances by which orchid are fertilised by insects, 2nd ed. John Murray, London. Dodson, C., Hills, H., 1966. Gas chromatography of orchid fragrances. Amer. Orchid Soc. Bull. 35, 720-725. Dodson, C.H., 1962a. The importance of pollination in the evolution of the orchids of tropical America. Amer. Orchid Soc. Bull. 31, 525-554, 641-649, 731-735. Dodson, C.H., 1962b. Pollination and variation in the subtribe Catasetinae (Orchidaceae). Ann. Mo. Bot. Gard. 49, 35-56.

Dodson, C.H., 1965a. Agentes de polinización y su Influencia en sobre la evolución de la familia Orquidacea. Universidad Nacional de la Amazonía Peruana. Instituto General de Investigaciones, Iquitos, Perú. Dodson, C.H., 1965b. Studies in orchid pollination: The genus Coryanthes. Amer. Orchid Soc. Bull. 34, 680-687. Dodson, C.H., 1966. Ethology of some bees of the tribe Euglossini (Hymenoptera: Apidae). J. Kans. Entomol. Soc. 39, 607-629. Dodson, C.H., 1967. Relationships between pollinators and orchid flowers. Atas do Simpósio sobre a Biota Amazônica 5, 1-72. Dodson , C.H., 1970. The role of chemicals attractants in orchid pollination, in: Chambers, K.L. (Ed.), Biochemical coevolution. Oregon State University Press, Corvallis, pp. 83-107. Dodson, C.H., 1978a. The catasetums (Orchidaceae) of Tapakuma, Guyana. Selbyana 2, 159168. Dodson, C.H., 1978b. Three new South American species of Catasetum (Orchidaceae). Selbyana 2, 156-158. Dodson, C.H., Dressler, R.L., Hills, H.G., Adams, R.M., Williams, N.H., 1969. Biologically active compounds in orchid fragrances. Science 164, 1234-1249. Dodson, C.H., Frymire, G.P., 1961a. Natural pollination of orchids. Mo. Bot. Gard. bull. 49, 133-152. Dodson, C.H., Frymire, G.P., 1961b. Preliminary studies in the genus Stanhopea (Orchidaceae). Ann. Mo. Bot. Gard. 48, 137-172. Doorn, W.G.V., 1997. Effects of pollination on floral attraction and longevity. J. Exp. Bot. 48, 1615-1622. Dötterl, S., Glück, U., Jürgens, A., Woodring, J., Aas, G., 2014. Floral reward, advertisement and attractiveness to honey bees in dioecious Salix caprea. Plos One 9, e93421.

Dötterl, S., Vereecken, J.N., 2010. The chemical ecology and evolution of bee-flower interactions: a review and perspectives. Can. J. Zool. 88, 668-697. Dressler, R.L., 1968. Observations on orchids and euglossine bees in Panama and Costa Rica. Rev. Biol. Trop. 15, 143-183. Dressler, R.L., 1978. New species of Euglossa from México and Central America. Rev. Biol. Trop. 26, 167-185. Dressler, R.L., 1979. Eulaema bombiformis, E. meriana, and müllerian mimicry in related species (Hymenoptera: Apidae). Biotropica 11, 144-151. Dressler, R.L., 1982. Biology of the orchid bees (Euglossini). Annu. Rev. Ecol. Syst. 13, 373394. Ducke, A., 1902. As especies paraenses do genero Euglossa. Bol. Mus. Para. Emílio Goeldi 3, 561-577. Eltz, T., Roubik, D.W., Lunau, K., 2005. Experience-dependent choices ensure speciesspecific fragrance accumulation in male orchid bees. Behav. Ecol. Sociobiol. 59, 149-156. Eltz, T., Whitten, W.M., Roubik, D.W., Linsenmair, E.K., 1999. Frangrance collection, storage, and accumulation by individual male orchid bees. J. Chem. Ecol. 25, 157-176. Eltz, T., Zimmermann, Y., Pfeiffer, C., Pech, J.R., Twele, R., Francke, W., Quezada-Euan, J.J.G., Lunau, K., 2008. An olfactory shift is associated with male perfume differentiation and species divergence in orchid bees. Curr. Biol. 18, 1844-1848. Frankie, G.W., Haber, W.A., Opler, P.A., Bawa, K.S., 1983. Characteristics and organization of the large bee pollination system in the Costa Rican dry forest, in: Jones, C.E., Little, R.J. (Eds.), Handbook of experimental pollination biology. Van Nostrand & Reinhold. 558pp., New York, pp. 411-447. Fulop, D., 2009. Biomechanics, pollination, and evolution of Catasetum (Catasetinae, Orchidaceae), Department of Organismic and Evolutionary Biology Harvard University, p. 164.

Gerlach, G., 2007. The true sexual life of Catasetum and Cycnoches. Caesiana 28, 57-62. Gerlach, G., Schill, R., 1989. Fragrance analyses, an aid to taxonomic relationships of the genus Coryanthes (Orchidaceae). Plant Syst. Evol. 168, 159-165. Gerlach, G., Schill, R., 1991. Composition of orchid scents attracting euglossine bees. Bot. Acta 104, 379-391. González, J., 1998. Associated plants and distribution of the orchid bee genus Eulaema (Apidae: Bombinae: Euglossini) in Venezuela. Boletín del Centro de Investigaciones Biológicas 32. Gori, D.F., 1983. Post-pollination phenomema and adaptative floral changes, in: Jones, C.E., Little, R.J. (Eds.), Handbook of experimental pollination biology. Van Nostrand Reinhold Company, Inc, New York, pp. 31-43. Govaerts, R., Bernet, P., Kratochvil, K., Gerlach, G., Carr, G., Alrich, P., Pridgeon, A.M., Pfahl, J., Campacci, M.A., Baptista, D.H., Tigges, H., Shaw, J., Cribb, P., George, A., Kreuz, K., Wood, J., 2015. World Checklist of Orchidaceae. Facilitated by the Royal Botanic Gardens, Kew. Available from: http://apps.kew.org/wcsp/ (accessed 27 October 2015). Gregg, K.B., 1975. The effect of light intensity on sex expression in species of Cycnoches and Catasetum (orchidaceae). Selbyana 1, 101-113. Gregg, K.B., 1982. Sunlight-Enhanced Ethylene Evolution by Developing Inflorescences of Catasetum and Cycnoches and Its Relation to Female Flower Production. Botanical Gazette 143, 466-475. Harrison, N., Kidner, C.A., 2011. Next-generation sequencing and systematics: what can a billion base pairs of DNA sequence data do for you? Taxon 60, 1552-1566. Heinrich, B., 1993. The hot-blooded insects. Strategies and mechanisms of thermoregulation. Harvard University Press, Cambridge, USA. Hetherington-Rauth, M.C., Ramírez, S.R., 2015. Evolutionary trends and specialization in the euglossine bee–pollinated orchid genus Gongora. Ann. Mo. Bot. Gard. 100, 271-299.

Hetherington-Rauth, M.C., Ramírez, S.R., 2016. Evolution and diversity of floral scent chemistry in the euglossine bee-pollinated orchid genus Gongora. Ann. Bot., mcw072. Hills, H., 1989. Fragrance cycling in Stanhopea pulla (Orchidaceae, Stanhopeinae) and identification of trans-limonene oxide as a major fragrance component. Lindleyana 4, 61-67. Hills, H.G., Williams, N.H., 1990. Fragrance cycle of Clowesia rosea. Orquídea (Méx.) 12, 19-22. Hills, H.G., Williams, N.H., Dodson, C.H., 1968. Identification of some orchid fragrance components. Amer. Orchid Soc. Bull. 37, 967-971. Hills, H.G., Williams, N.H., Dodson, C.H., 1972. Floral fragrances and isolating mechanisms in the genus Catasetum (Orchidaceae). Biotropica 4, 61-76. Hills, H.G., Williams, N.H., Whitten, W.M., 1999. Frangrance of catasetums, in: Holst, A.W. (Ed.), The world of catasetums. Timber Press Inc., Portland, pp. 263-272. Hoehne, F.C., 1933. Contribuição para o conhecimento do gênero Catasetum L. C. Rich e especialmente o hermafroditismo e trimorfismo de suas flores. Boletim de Agricultura, 133196. Holst, A.W., 1999. The world of catasetums. Timber Press, Portland. Janzen, D.H., 1981a. Bee arrival at two Costa-Rican female Catasetum orchid inflorescences and a hypothesis on euglossine population structure. Oikos 36, 177-183. Janzen, D.H., 1981b. Differential visitation of Catasetum orchid male and female flowers. Biotropica 13, 77. Johnson, S., 2006. Pollinator-driven speciation in plants, in: Harder, L.D., Barrett, C.H. (Eds.), Ecology and evolution of flowers. Oxford University Press, Oxford, UK, pp. 295–310. Kaiser, R., 1993. The scent of orchids - olfactory and chemical investigation. Elsevier, Amsterdam. Kaiser, R., 2011. Scent of the Vanishing Flora. Wiley-VCH, Zurich.

Knudsen, J.T., Eriksson, R., Gershenzon, J., Stahl, B., 2006. Diversity and distribution of floral scent. Bot. Rev. 72, 1-120. Lindquist, N., A. Battiste, M., Mark Whitten, W., H. Williams, N., Strekowski, L., 1985. Trans-carvone oxide, a monoterpene epoxide from the fragrance of Catasetum. Phytochemistry 24, 863-865. Lopes, A.V., Machado, I.C., Aguiar, A.V., Rebêlo, J.M.M., 2007. A scientific note on the occurrence of Euglossini bees in the Caatinga, a brazilian tropical dry forest. Apidologie 38, 472–473. Mansfeld, R., 1932. Die Gattung Catasetum LC Rich. Repert. Nov. Spec. Regni Veg. 31, 99125. Mardis, E.R., 2008. The impact of next-generation sequencing technology on genetics. Trends Genet. 24, 133-141. Martini, P., Schlindwein, C., Montenegro, A., 2003. Pollination, flower longevity, and reproductive biology of Gongora quinquenervis Ruíz & Pavón (Orchidaceae) in an atlantic forest fragment of Pernambuco, Brazil. Plant Biol. 5, 495-503. Milet-Pinheiro, P., Navarro, D.M.A.F., Dötterl, S., Carvalho, A.T., Pinto, C.E., Ayasse, M., Schlindwein, C., 2015. Pollination biology in the dioecious orchid Catasetum uncatum: How does floral scent influence the behaviour of pollinators? Phytochemistry 116, 149-161. Mitko, L., Weber, M.G., Ramirez, S.R., Hedenström, E., Wcislo, W.T., Eltz, T., 2016. Olfactory specialization for perfume collection in male orchid bees. The Journal of Experimental Biology 219, 1467-1475. Moore, M.J., Dhingra, A., Soltis, P.S., Shaw, R., Farmerie, W.G., Folta, K.M., Soltis, D.E., 2006. Rapid and accurate pyrosequencing of angiosperm plastid genomes. BMC Plant Biol. 6, 17. Murren, C.J., 2002. Effects of habitat fragmentation on pollination: pollinators, pollinia viability and reproductive success. J. Ecol. 90, 100-107.

Oliveira, M.L., 2000. O Gênero Eulaema Lepeletier, 1841 (Hymenoptera, Apidae, Euglossini): Filogenia, Biogeografía e Relações com as Orchidaceae. Universidade de São Paulo, Ribeirão Preto, Brasil. Parks, M., Cronn, R., Liston, A., 2009. Increasing phylogenetic resolution at low taxonomic levels using massively parallel sequencing of chloroplast genomes. BMC Biology 7, 84. Pawliszyn, J., 2002. Chapter 13 Solid phase microextraction, Comprehensive Analytical Chemistry. Elsevier, pp. 389-477. Peakall, R., Ebert, D., Poldy, J., Barrow, R.A., Francke, W., Bower, C.C., Schiestl, F.P., 2010. Pollinator specificity, floral odour chemistry and the phylogeny of Australian sexually deceptive Chiloglottis orchids: implications for pollinator-driven speciation. New Phytol. 188, 437-450. Pereboom, J.J.M., Biesmeijer, J.C., 2003. Thermal constraints for stingless bee foragers: the importance of body size and coloration. Oecologia 137, 42-50. Peruquetti, R.C., Campos, L.A.O., Coelho, C.D.P., Abrantes, C.V.M., Lisboa, L.C.O., 1999. Abelhas Euglossini (Apidae) de áreas de Mata Atlântica: abundância, riqueza e aspectos biológicos. Rev. Bras. Zool. 16, 101-118. Petini-Benelli, A., 2012. Orquídeas de Mato Grosso - Genus Catasetum L.C. Rich ex. Kunth. PoD Editora, Rio de Janeiro. Pokorny, T., Hannibal, M., Quezada-Euan, J.J.G., Hedenström, E., Sjöberg, N., Bång, J., Eltz, T., 2013. Acquisition of species-specific perfume blends: influence of habitat-dependent compound availability on odour choices of male orchid bees (Euglossa spp.). Oecologia 172, 417-425. Porsch, O., 1955. Zur Biologie der Catasetum-Blüte. Österr. Bot. Z. 102, 117-157. Pridgeon, A., Chase, M., 1998. Phylogenetics of subtribe Catasetinae (Orchidaceae) from nuclear and chloroplast DNA sequences, Proc. 15th World Orchid Conference. Naturalia Publications, Turriers, France, pp. 275-281.

Raguso, R.A., 2004a. Flowers as sensory billboards: progress towards an integrated understanding of floral advertisement. Curr. Opin. Plant Biol. 7, 434-440. Raguso, R.A., 2004b. Why do flowers smell? - The chemical ecology of fragrance-driven pollination, in: Cardé, R.T., Millar, J.G. (Eds.), Advances in Insect Chemical Ecology. Cambridge University Press, Cambridge, pp. 151-178. Raguso, R.A., 2008. Wake up and smell the roses: the ecology and evolution of floral scent. Annu. Rev. Ecol. Evol. Syst. 39, 549-569. Ramirez, S., Dressler, R.L., Ospina, M., 2002. Abejas euglosinas (Hymenoptera: Apidae) de la région neotropical: listado de especies con notas sobre su biología. Biota Colombiana 3, 7118. Ramírez, S.R., Eltz, T., Fujiwara, M.K., Gerlach, G., Goldman-Huertas, B., Tsutsui, N.D., Pierce, N.E., 2011. Asynchronous diversification in a specialized plant-pollinator mutualism. Science 333, 1742-1746. Rocha-Filho, L.C., Krug, C., Silva, C.I., Garófalo, C.A., 2012. Floral resources used by Euglossini bees (Hymenoptera: Apidae) in coastal ecosystems of the Atlantic Forest. Psyche 2012, 1-13. Rolfe, R.A., 1890. On the Sexual Forms of Catasetum, with special reference to the Researches of Darwin and others. J. Linn. Soc. Lond. Bot. 27, 206-225. Romero, G., 1990. Phylogenetic relationships in subtribe Catasetinae (Orchidaceae, Cymbidieae). Lindleyana 5, 160-181. Romero, G.A., Carnevali Fernández-Concha, G., Pridgeon, A.M., 2009. Catasetum, in: Pridgeon, A.M., Cribb, P.J., Chase, M.W., Rasmussen, F.N. (Eds.), Genera Orchidacearum. Volume 5. Epidendroideae (Part two). Oxford University Press, Oxford, pp. 22-25. Romero, G.A., Jenny, R., 1993. Contributions toward a monograph of Catasetum (Catasetinae, Orchidaceae) I: A checklist of species, varieties, and natural hybrids. Harv. Pap. Bot., 59-84.

Romero, G.A., Nelson, C.E., 1986. Sexual dimorphism in Catasetum orchids: forcible pollen emplacement and male flower competition. Science 232, 1538-1540. Roubik, D.W., 1989. Ecology and natural history of tropical bees. Cambridge University Press, New York. Roubik, D.W., Ackerman, J.D., 1987. Long-term ecology of euglossine orchid-bees (Apidae: Euglossini) in Panama. Oecologia 13, 321-332. Roubik, D.W., Hanson, P.E., 2004. Orchid bees: biology and field guide. INBIO, San Jose, Costa Rica. Sazima, M., Vogel, S., Cocucci, A., Hausner, G., 1993. The perfume flowers of Cyphomandra (Solanaceae) - pollination by euglossine bees, bellows mechanism, osmophores, and volatiles. Plant Syst. Evol. 187, 51-88. Schiestl, F.P., 2010. The evolution of floral scent and insect chemical communication. Ecol. Lett. 13, 643-656. Schiestl, F.P., Ayasse, M., Paulus, H.F., Löfstedt, C., Hansson, B.S., Ibarra, F., Francke, W., 1999. Orchid pollination by sexual swindle. Nature 399, 421-422. Schiestl, F.P., Dötterl, S., 2012. The evolution of floral scent and olfactory preferences in pollinators: coevolution or pre-existing bias? Evolution 66, 2042-2055. Schiestl, F.P., Peakall, R., Mant, J., Ibarra, F., Schulz, C.M., Franke, S., Francke, W., 2003. The chemistry of sexual deception in an orchid-wasp pollination system. Science 302, 437438. Soler, C.C.L., Proffit, M., Bessière, J.-M., Hossaert-McKey, M., Schatz, B., Irwin, R., 2012. Evidence for intersexual chemical mimicry in a dioecious plant. Ecol. Lett. 15, 978-985. Tholl, D., Röse, U.S.R., 2006. Detection and Identification of Floral Scent Compounds, in: Dudareva, N., Pichersky, E. (Eds.), Biology of Floral Scent. CRC Press, Boca Raton, pp. 126.

van der Pijl, L., Dodson, C.H., 1969. Orchid flowers: their pollination and evolution. University of Miami Press, Coral Gables. Vogel, S., 1966. Parfümsammelnde Bienen als Bestäuber von Orchidaceen und Gloxinia. Österr. Bot. Z. 113, 302–361. Vogel, S., 1990. The role of scent glands in pollination: on the structure and function of osmophores. Smithsonian Institution Libraries/National Science Foundation, Washington, DC. Weber, M.G., Mitko, L., Eltz, T., Ramírez, S.R., Scherber, C., 2016. Macroevolution of perfume signalling in orchid bees. Ecol. Lett. Whitten, W.M., Hills, H.G., Williams, N.H., 1988. Occurrence of ipsdienol in floral fragrances. Phytochemistry 27, 2759-2760. Whitten, W.M., Williams, N.H., 1992. Floral fragrances of Stanhopea (Orchidaceae). Lindleyana 7, 130-153. Whitten, W.M., Williams, N.H., Armbruster, W.S., Battiste, M.A., Strekowski, L., Lindquist, N., 1986. Carvone oxide: an example of convergent evolution in euglossine pollinated plants. Syst. Bot. 11, 222-228. Williams, N.H., 1983. Floral fragrances as cues in animal behavior, in: Jones, E.C., Little, R.J. (Eds.), Handbook of experimental pollination biology. Van Nostrand Reinhold, New York, pp. 50-72. Williams, N.H., Dodson, C.H., 1972. Selective attraction of male euglossine bees to orchid floral fragrances and its importance in long distance pollen flow. Evolution 26, 84-95. Williams, N.H., Whitten, M., 1999. Molecular phylogeny and floral fragrances of male euglossine bee-pollinated orchids: a study of Stanhopea (Orchidaceae). Plant Species Biol. 14, 129-136. Williams, N.H., Whitten, W.M., 1983. Orchid floral fragrances and male euglossine bees: methods and advances in the last sesquidecade. Biol. Bull. 164, 355-395.

Yang, J.-B., Tang, M., Li, H.-T., Zhang, Z.-R., Li, D.-Z., 2013. Complete chloroplast genome of the genus Cymbidium: lights into the species identification, phylogenetic implications and population genetic analyses. BMC Evol. Biol. 13, 1-12. Zimmermann, Y., Ramírez, S.R., Eltz, T., 2009. Chemical niche differentiation among sympatric species of orchid bees. Ecology 90, 2994-3008. Zucchi, R., Sakagami, S.F., Camargo, J.M.F., 1969. Biological observations on a Neotropical parasocial bee, Eulaema nigrita, with a review on the biology of Euglossinae (Hymenoptera, Apidae). A comparative study. J. Fac. Sci. Hokkaido Univ. Zool. 17, 271-380.

Fig. 1. Overview of the sexual dimorphism (A, B) and morphological traits used for infrageneric classification in the genus Catasetum (B-D). Female (A) and male flowers (B) of Catasetum juruenense. Note the position of the lip above and below the column in the female and male flowers, respectively. Arrows indicate the rudimentary antennae in flowers of Catasetum (subgen. Pseudocatasetum) longifolium (C), the symmetric antennae (section Isoceras) of Catasetum (subgen. Catasetum) juruenense (B) and the asymmetric antennae (section Catasetum) in flowers of Catasetum (subgen. Catasetum) osculatum (D). All photographs by Juan Fernández Gómez, except for (A) by Günter Gerlach.

Fig. 2. New reports on pollinators of Catasetum species (A-K). Pollination by Euglossa (AG): C. globiflorum (A), C. parguazense (B), C. schunkei (C), C. semicirculatum (D), C. denticulatum (E), C. juruenense (F), C. pulchrum (G) and C. sanguineum (H; female flower). Pollination by Eulaema (I-K): C. schmiditianum (I), C. tabulare (J) and C. x faustoi (K). Note the pollinarium attached to the thorax of the Euglossa males in photograph G and H. Photographs B, D-K, were made in the state of Rondônia (Brazil) within the natural range of these species. The only exception is C. tabulare that occurs in Colombia, where species of Eulaema are also the pollinators. Photographs A and C were made in a natural population in

Rio de Janeiro (Brazil) and Panguana (Peru), respectively. Photographs by Juan Fernandéz Gómez (D-K), by Valdir Sanches (B), by Luiz Valter (A), and by Günter Gerlach (C).

Fig. 3. CAP plot analyzing the floral fragrances of 23 Catasetum species pollinated by Eulaema, Euglossa and Eufriesea, based on semi-quantitative Bray-Curtis similarities. Species for which fragrance profiles were only partially identified (less than 70%) are not included (see supplementary material Table S2). Names of species on sections Isoceras and Anisoceras (subgenus Catasetum) are in blue and red, respectively. Names of species of subgenus Pseudocatasetum are in black.

Table 1. List of Catasetum species for which floral visitors have been reported and floral scent chemistry has been characterized. (*) indicates species for which floral scents have not been satisfactorily characterized (less than 70% of the whole scent bouquet). Letters represent the role of floral visitors: effective pollinator (P), pollinarium carrier (PC), unknown (U), non-

pollinator (NP). Numbers represent the literature citations. Effective pollinators are those species that carry pollinarium and visit both male and female flowers. Species/subgenus/section

Visitor/Pollinator

Scent characterization

Catasetum collare Cogn. (1895)

?

Yes [4,7,14]

Catasetum expansum Rchb.f. (1878)

Yes [4,7,14,19,22]

Catasetum gnomus Linden & Rchb.f. (1873)

El. bomboides (Friese, 1923) P[4], El. cingulata (Fabricius, 1804)P[4, 10, 20], El. polychroma (Mocsáry, 1899)P[4, 20, 21] ?

Catasetum incurvum Klotzsch (1854)

Eulaema sp. ††† P[2]

No

Catasetum integerrimum Hook. (1840)

Yes [4,14,26]

Catasetum laminatum Lindl. (1840)†

El. cingulataP[4], El. polychromaP[4], El. meriana (Olivier, 1789)P[26], Exaerete frontalis (GuérinMéneville, 1845)P[26] ?

Catasetum × faustoi Bicalho (1996)

Eulaema sp. ††† P[1,2]

No

Catasetum macrocarpum Rich. ex Kunth (1822)†

No [4,7,14]

Catasetum osculatum Lacerda & Castro (1995)

El. seabrai bennettii Moure, 1967P[4], El. bombiformis (Packard, 1869)P[5], El. cingulataP[4, 16], El. merianaP[4], El. seabrai mimetica Moure, 1967P[4], El. nigrita Lepeletier, 1841P[27], El. peruviana (Friese, 1903)P[16], El. seabrai Moure, 1960P[27], El. seabrai luteola Moure, 1967U[28], El. terminata Smith, 1874P[4], Eg. ignita Smith, 1874U[10], Eg. imperialis Cockerell, 1922NP[27], Eg. piliventris Guérin-Méneville, 1845U[27] El. bomboidesP[4,20], El. cingulataP[4,20], El. polychromaP[4,20], El. speciosa (Mocsáry, 1897)P[20] El. cingulataP[4], El. polychromaP[4,29,30], El. merianaP[30], Eg. gorgonensis erythrophana Dressler, 1978U[31], Eg. flammea Moure, 1969U[10] Eulaema cingulataP[35]

Catasetum pendulum Dodson (1977)†

El. polychromaP[10,36]

No

Catasetum pileatum Rchb.f. (1882)

Yes [4,7,14,19]

Catasetum schmidtianum Miranda & Lacerda (1992)

El. bombiformisP[37], El. cingulataP[4], El. merianaP[38] El. nigritaP[38], El. peruvianaP[4,19], El. seabraiP[38], El. seabrai mimeticaU[28] Ef. violacens (Mocsáry, 1898)P[41], Eg. augaspis Dressler, 1982NP[29], Eg. chalybeata Friese, 1925PC[18], Eg. cordata (Linnaeus, 1758)U[27], Eg. ignitaNP[10,29], Eg. imperialisPC[18], El. cingulataPC[4] Eulaema cf. cingulata ††† P[1]

Catasetum tabulare Lindl. (1844)

El. cingulataP[4]

Yes [4,7,14]

Catasetum viridiflavum Hook. (1843).

El. cingulataP[4], El. nigritaP[12,45], El. marcii Nemésio, 2009P[12], Ex. frontalisU[10,18]

Yes [4,7,14,19,46]

Catasetum ariquemense Miranda & Lacerda (1992)

Euglossa sp. ††† P[1,2]

No

Catasetum atratum Lindl. (1838)

Euglossa sp. ††† P[3]

Yes [4]

Subgenus Catasetum section Anisoceras

Catasetum macroglossum Rchb.f. (1877)

Catasetum maculatum Kunth (1822)

Catasetum saccatum Lindl. (1840)

Yes[4,14]

No [7]

Yes [4,7,14,22] Yes [4,7,14,32]

No

Yes [22]

No

Subgenus Catasetum section Isoceras

Catasetum callosum Lindl. (1840)*

Eg. augaspisP[5], Eg. cognata Moure, 1970P[5], Eg. Yes [4,7,8] cordataP[4,6], Eg. mixta Friese, 1899PC[5], El. cingulataNP[5] Eg. allosticta Moure, 1969PC[9,10], Eg. cognata No [4] PC[9], Eg. cordataPC[6], Eg. cyanaspis Moure, 1968P[11], Eg. cybelia Moure, 1968PC[12], Eg. deceptrix Moure, 1968PC[4,10], Eg. dissimula Dressler 1978PC[9], Eg. dodsoni Moure 1965PC[12], Eg. gorgonensis Chessman, 1929PC[4], Eg. hemichlora Cockerell, 1917U[10], Eg. heterosticta Moure, 1968PC[12], Eg. mixta Friese, 1899PC[12], Eg. tridentata Moure, 1970P[10,11,12], Eg. variabilis Friese, 1899P[4, 10, 12], El. merianaNP[10,12,13], El. nigritaU[10] ? Yes [4,14]

Catasetum cernuum (Lindl.) Rchb.f. (1863)

Ef. violacea (Blanchard, 1840)P[15]

No

Catasetum cirrhaeoides Hoehne (1915)*

?

Yes [4,14]

Catasetum complanatum Miranda & Lacerda (1992)

Euglossa sp. ††† P[1]

No

Catasetum confusum Romero (1993)

Euglossa sp. ††† P[1]

No

Catasetum cristatum Lindl. (1825)*

?

Yes [7]

Catasetum denticulatum Miranda (1986)

Euglossa sp. ††† P[1,2]

No

Catasetum ferox Kraenzl. (1895)

Euglossa sp. ††† P[1]

No

Catasetum fimbriatum (C.Morren) Lindl. (1850)

Yes [4,22]

Catasetum gladiatorium Lacerda (1998).

Ef. auriceps Friese, 1899P[4], Ef. combinata (Mocsáry, 1897)P[18], El. cingulataPC[23] Euglossa P[1]

Catasetum globiflorum Hook. (1842)

Euglossa sp.P[24]

No

Catasetum hookeri Lindl. (1824)

Eg. cordataP[4], Eg. stellfeldi Moure, 1947P[25]

No

Catasetum hopkinsonianum Carr & Castro (2008)

Euglossa sp. ††† P[1]

No

Catasetum juruenense Hoehne (1915)

Euglossa sp. ††† P[1]

No

Catasetum labiatum Barb.Rodr. (1882)*

?

Yes [7]

Catasetum lemosii Rolfe (1894)*

?

Yes [7]

Catasetum luridum Lindl. (1833)

Eg. cordataP[4,27]

Yes [4]

Catasetum maranhense Lacerda & Silva (1998)

Euglossa sp. ††† P[1]

No

Catasetum micranthum Barb.Rodr. (1882)

?

Yes [7]

Catasetum microglossum Rolfe (1913)

?

Yes [4,14]

Catasetum napoense Dodson (1978)

?

Yes [7,33]

Catasetum ochraceum Lindl. (1844)*

Yes [4]

Catasetum parguazense Romero & Carnevali (1989)

Eg. gaianii Dressler, 1982P[34], Eg. modestior Dressler, 1982P[34] Euglossa sp. ††† P[2]

Catasetum planiceps Lindl. (1843)

El. cingulataP[39]

Yes [7]

Catasetum pulchrum N.E.Br. (1887)

Euglossa sp. ††† P[1]

No

Catasetum purum Nees & Sinning (1824)

Euglossa sp.P[24]

Yes [7,33]

Catasetum barbatum (Lindl.) Lindl.(1844)

Catasetum bicolor Klotzsch (1854)*††

No

No

Catasetum reichenbachianum Mansf. (1930)

Eg. augaspisP[4,40]

No

Catasetum rondonense Pabst (1967)

Euglossa sp. ††† P[1]

No

Catasetum sanguineum Lindl. & Paxton (1851)

Eg. variabilis Friese, 1899P[42]

No

Catasetum schunkei Dodson & Benn. (1989)

Euglossa sp.P[43]

No

Catasetum semicirculatum Miranda (1986)

Euglossa sp. ††† P[1]

No

Catasetum socco (Vell.) Hoehne (1952)

Eg. stellfeldiP[25]

No

Catasetum tenebrosum Kraenzl. (1910)

Euglossa sp. ††† P[1]

Yes [4,14]

Catasetum thompsonii Dodson (1978)

Eg. augaspisP[6], Eg. cognataP[6,16], Eg. cordataP[6,16], Eg. liopoda Dressler, 1982P[6,16], Eg. mixta Friese, 1899P[6,16], El. cingulataNP[16] Eg. carolina Nemésio 2009P[44], Eg. nanomelanotricha Nemésio 2009P[44]

No

El. bombiformisP[16], El. bomboidesU[17], El. cingulataP[4, 16], El. merianaP [4,11,16], El. nigritaP[10,18], Eg. ignitaNP[10,16] El. bombiformisP[5], El. cingulataP[4,16], El. merianaP[4], El. peruvianaP[5]

Yes [4,7,14,19]

Catasetum uncatum Rolfe (1895)

Yes [44]

Subgenus Pseudocatasetum Catasetum discolor (Lindl.) Lindl. (1844)

Catasetum longifolium Lindl. (1839)

Yes [4,14,19,22]

† Characterization of floral scents of C. macrocarpum, C. pendulum and C. laminatum is not given individually by Hills et al. (1972; 1999), but rather together with many other species belonging to what they called "C. maculatum complex". †† Samples of C. bicolor were collected, but no compound was detected with gas chromatography by Hills et al. (1972). ††† Personal communication with Gómez, Sanches and Varela (see below) was made after finding photographs of euglossine bees visiting flowers of Catasetum on the internet forum "Catasetum Brasil" (https://www.facebook.com/groups/catasetumbrasil/?ref=bookmarks). Original photographs in high resolution were then provided by the picture owners (see figure 3).

(1) Gómez (pers. comm.), (2) Sanches (pers. comm.), (3) Varela (pers. comm.), (4) Hills et al. (1972), (5) Williams and Dodson (1972), (6) Dodson (1978b), (7) Hills et al. (1999), (8) Kaiser (2011), (9) Roubik and Ackerman (1987), (10) Roubik and Hanson (2004), (11) Dressler (1968), (12) Ackerman (1983), (13) Dressler (1979), (14) Hills et al. (1968), (15) Hoehne (1933), (16) Dodson (1978a), (17) Dodson and Frymire (1961a), (18) Ramirez et al. (2002), (19) Whitten et al. (1986), (20) Dodson (1962b), (21) Dodson (1966), (22) Gerlach and Schill (1991), (23) Peruquetti et al. (1999), (24) Milet-Pinheiro (unpubl. data), (25) Rocha-Filho et al. (2012), (26) Cancino and Damon (2007), (27) Dodson (1967), (28) Oliveira (2000), (29) Dodson (1965a), (30) Janzen (1981a), (31) Dressler (1978), (32) Lindquist et al. (1985), (33) Whitten et al. (1988), (34) Romero and Nelson (1986), (35) Petini-Benelli (2012), (36) Holst (1999), (37) González (1998), (38) Dodson and Hills (1966), (39) Romero et al. (2009), (40) Dressler (1982), (41) Zucchi et al. (1969), (42) Gerlach (2007), (43) Gerlach (unpubl. data), (44) Milet-Pinheiro et al. (2015), (45) Ackerman and Roubik (2012), (46) Kaiser (1993).

Table 2. Number and percentage amount of compounds (contribution of each compound area relative to the total) in the floral scent chemistry of Catasetum species. Compounds are listed alphabetically within compound classes. Only compounds that contributed ≥ 1% of the total scent discharge in at least one of the species are shown. Species for which floral scent chemistry was only partially identified (less than 70% of the total) are not included here. Abbreviations: Catasetum atratum (ATR), C. barbatum (BAR), C. collare (COL), C. discolor (DIS), C. expansum (EXP), C. fimbriatum (FIM), C. gnomus (GNO), C. integerrimum (INT), C. longifolium (LON), C. luridum (LUR), C. macroglossum (MAC), C. maculatum (MAU), C. micranthum (MIC), C. microglossum (MCR), C. napoense (NAP), C. pileatum (PIL), C. purum (PUR), C. saccatum (SAC), C. tabulare (TAB), C. tenebrosum (TEN), C. tuberculatum (TUB), C. uncatum (UNC), and C. viridiflavum (VIR). A detailed list with all compounds detected in the species revised here, as well as the references thereof, is given as supplementary data, Table S2. ATR

BAR COL

DIS

EXP

FIM

GNO INT

LON LUR

MAC MAU MIC

MCR NAP

PIL

PUR

SAC

TAB

TEN

TUB

UNC VIR

3

33

5

10

14

5

12

30

14

2

15

6

1

4

2

21

2

10

6

2

2

74

35

Benzyl acetate

-

-

-

20

14

1

-

tr

9

-

7

-

-

-

-

tr

-

-

-

-

-

-

14

Benzyl benzoate

-

-

-

-

-

-

-

-

2

-

-

-

-

-

-

-

-

-

-

-

-

tr

3

Chavicol

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

1

-

p-Cymene

-

-

-

1

9

-

-

1

2

-

8

2

-

-

-

4

-

-

9

-

-

tr

6

1,2-Dimethoxybenzene

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

16

-

1,4-Dimethoxybenzene

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

42

-

-

Dimethyl Styrene

-

-

-

-

4

-

-

tr

-

-

2

-

-

-

-

-

-

-

-

-

-

-

2

Estragol

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

3

-

Guaiacol

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

7

-

Methyl benzoate

-

-

32

tr

-

-

3

tr

-

-

-

-

-

-

-

-

-

-

-

-

-

tr

-

(E)-Methyl cinnamante (E)-Methyl-pmethoxycinnamate

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

7

-

-

-

-

-

tr

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

3

-

Number of compounds Aromatics

(Z)-Methyl-pmethoxycinnamate Methyl salicylate

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

2

-

-

-

64

-

-

-

44

-

-

-

-

-

-

-

-

-

-

-

-

-

-

1

-

Camphene

-

-

-

1

1

-

-

tr

-

-

1

3

-

-

-

1

-

-

3

-

-

-

-

Carvone

-

-

-

7

3

12

-

4

6

-

2

-

-

-

-

6

-

-

5

-

-

tr

5

1,8-Cineole

-

Tr

1

2

7

-

7

10

5

84

2

4

-

5

-

11

-

18

-

-

34

24

2

(E)-Carvone epoxide

-

-

-

15

35

-

3

42

17

-

23

74

-

-

-

47

-

12

66

-

-

-

57

(E)-Dihydrocarvone

-

-

-

2

2

-

tr

tr

1

-

2

5

-

-

-

1

-

4

3

-

-

tr

1

(E)-limonene epoxide

-

-

-

-

3

-

5

3

2

-

2

-

-

-

-

2

-

3

-

-

-

tr

1

(E)-Ocimene

-

1

-

-

-

-

-

-

-

-

-

-

-

84

-

-

-

-

5

-

-

tr

-

Geraniol

-

-

-

-

-

75

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Ipsdienol

-

-

-

-

-

-

-

-

-

-

-

-

-

-

36

-

41

-

-

47

-

tr

-

Limonene

-

tr

-

-

1

-

2

-

3

-

2

-

-

-

-

1

-

4

-

-

-

7

tr

Myrcene

90

tr

-

-

-

1

-

tr

-

-

-

-

-

-

34

-

42

-

-

47

-

13

-

α-Phellandrene

-

-

-

-

1

1

1

tr

1

-

1

-

-

-

-

2

-

2

-

-

-

-

-

α-Pinene

2

1

tr

36

11

-

13

24

34

11

32

3

-

1

-

5

-

40

-

-

-

2

tr

β-Phellandrene

-

-

-

-

2

-

3

3

3

-

8

-

-

-

-

3

-

10

-

-

-

tr

-

β-Pinene

tr

-

tr

1

-

-

1

1

1

-

2

-

-

tr

-

tr

-

2

-

-

-

tr

tr

Sabinene

-

-

-

-

1

-

1

1

1

-

2

-

-

-

-

tr

-

2

-

-

-

1

tr

-

1

-

-

-

-

-

tr

-

-

-

-

100

-

-

-

-

-

-

-

-

3

2

(E)-β-Caryophyllene

-

4

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

10

-

(E)-α-Bergamotene

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

2

-

Bicyclogermacrene

-

2

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

α-Cubebene

-

2

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

β-Cubebene

-

1

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Cubebol

-

1

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

β-Elemene

-

3

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

tr

-

(E)-β-Farnesene Germacra-1(10), 5-dien4-ol

-

3

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

tr

-

-

60

-

-

-

-

-

-

-

-

-

-

-

-

-

1

-

-

-

-

-

-

-

Monoterpenes

N-bearing compounds Indole Sesquiterpenes

Germacrene A

-

9

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Germacrene D

-

7

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Humulene

-

3

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

tr

-

Total (%)

92

100

98

84

93

90

84

92

85

95

95

90

100

90

71

91

83

97

92

94

76

99

92

Total represents the sum of the relative percentage of compounds identified in the scent chemistry, including minor compounds not listed here (see Table S2, supplementary material). Tr - trace amount (percentage < 1%).

Table 3. List of species of Eulaema and Euglossa attracted to the six compounds explaining most of the chemistry dissimilarity (75%) between fragrances of species pollinated by Eulaema and by Euglossa. Asterisk (*) indicates species to which compounds are highly attractive. For references, see Ramírez et al. (2002). Compound Benzyl acetate

Euglossa species Eg. amazonica*, Eg. asarophora, Eg. augaspis*, Eg. chalybeata, Eg. cognata, Eg. crassipunctata, Eg. cybelia*, Eg. deceptrix, Eg. dissimula, Eg. dodsoni*, Eg. dressleri, Eg. flammea, Eg. hemichlora, Eg. heterosticta, Eg. ignita, Eg. imperialis, Eg. magnipes, Eg. modestior, Eg. mourei, Eg. sapphirina, Eg. tridentata, Eg. turbinifex, Eg. variabilis*, Eg. viridifrons*, Eg. viridissima

Eulaema species El. boliviensis, El. bombiformis*, El. cingulata, El. meriana*, El. mocsaryi*, El. nigrita, El. peruviana, El. polychroma, El. sororia

(E)-Carvone epoxide

Eg. cybelia, Eg. flammea, Eg. gorgonensis, Eg. ignita

El. bombiformis, El. cingulata, El. meriana, El. polychroma, El. speciosa

1,8-Cineole

Eg. alleni, Eg. allostica*, Eg. amazonica, Eg. analis, Eg. annectans*, Eg. asarophora, Eg. atroventa, Eg. augaspis*, Eg. azureoviridis, Eg. bidentata*, Eg. bursigera*, Eg. carinilabris, Eg. chalybeata*, Eg. championi*, Eg. charapensis, Eg. chlorina, Eg. cognata*, Eg. cordata*, Eg. crassipunctata, Eg. cyanaspis*, Eg. cybelia*, Eg. deceptrix, Eg. despecta*, Eg. dissimula*, Eg. dodsoni*, Eg. dressleri*, Eg. erythrochlora, Eg. fimbriata*, Eg. flammea*, Eg. gaianii, Eg. gibbosa, Eg. gorgonensis*, Eg. hansoni*, Eg. hemichlora*, Eg. heterosticta*, Eg. hyacinthina, Eg. ignita*, Eg. igniventris*, Eg. imperialis*, Eg. intersecta*, Eg. ioprosopa, Eg. iopyrrha*, Eg. leucotricha, Eg. liopoda*, Eg. macrorhyncha, Eg. maculilabris*, Eg. magnipes*, Eg. mandibularis, Eg. melanotricha, Eg. micans, Eg. mixta*, Eg. modestior*, Eg. mourei*, Eg. nigropilosa, Eg. obtusa, Eg. oleolucens, Eg. piliventris*, Eg. pleosticta*, Eg. prasina, Eg. purpurea*, Eg. rugilabris, Eg. sapphirina*, Eg. securigera, Eg. stillnonota*, Eg. townsendi*, Eg. tridentata*, Eg. trinotata, Eg. truncata*, Eg. turbinifex, Eg. ultima, Eg. variabilis*, Eg. villosiventris, Eg. violaceifrons, Eg. viridifrons, Eg. viridissima*

El. bombiformis, El. cingulata, El. meriana*, El. mocsaryi, El. nigrita*, El. polychroma*, El. speciosa

Ipsdienol

Eg. crassipunctata, Eg. cyanaspis, Eg. cyanura*, Eg. despecta, Eg. flammea, Eg. ignita, Eg. tridentata, Eg. viridis*

El. nigrita

Myrcene

Eg. asarophora, Eg. bidentata, Eg. championi, Eg. cyanaspis, Eg. despecta, Eg. heterosticta, Eg. ignita, Eg. tridentata, Eg. villosa

α-Pinene† † only the (-) isomer was shown to be attractive.

El. nigrita