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For most insects, just like for the majority of animals across phyla, the ability to detect a diversity of airborne molecules in their environment is critically important for survival (Hildebrand and Shepherd, 1997; Hansson and Stensmyr, 2011). Olfaction plays a pivotal role in the detection of food, hosts, predators, and kin (Olsson and Hansson, 2013), as well as in the attraction, location and identification of potential mates (Birch and Haynes, 1982; Cardé and Baker, 1984; Roelofs, 1984; Ayasse et al., 2001). The importance of olfaction in insects is apparent by looking at the elaborate antennal structures that exist in a diversity of shapes (Hansson and Stensmyr, 2011). Insect antennae are covered with different types of olfactory sensilla (Schneider and Steinbrecht, 1968), which contain the sensitive dendrites of the olfactory sensory neurons (Zacharuk, 1980; Couto et al., 2005). Olfactory stimulation occurs when odor molecules enter through pores or slits on the antenna surface (Steinbrecht, 1997) and are directed by odorant binding proteins (OBPs) that bind together with the volatile to olfactory receptors (ORs) situated in the membrane of these dendrites (Hallem and Carlson, 2006; Robertson and Wanner, 2006). These olfactory receptors vary in the type of molecules that activate them, their chemical tuning spectrum and the molecular receptive ranges (Hallem and Carlson, 2006; Getahun et al., 2013). Therefore, the olfactory periphery plays an important role in compound discrimination and represents the first step of specificity in olfactory sensitivity (Shields and Hildebrand, 2001; Hallem and Carlson, 2006; Brand et al., 2015) prior higher-level neural processing in the insects brain (see e.g., Renou, 2014).

Specificity in olfactory signals can be achieved either through complex molecules that are rare in nature (Chow and Wang, 1981; Ayasse et al., 2003; Schäffler et al., 2015) or by specific blends of relatively simple and ubiquitous compounds (Knudsen et al., 2006; Ayasse et al., 2011; Ayasse and Dötterl, 2014). While most insects synthesize such specific olfactory signals (e.g., pheromones) de novo or modify precursors found in their diet (Roelofs, 1984), male euglossine bees (Apidae, Euglossini) are known to harvest volatile compounds directly from flowers (Vogel, 1966; Dodson et al., 1969) as well as from non-floral sources (e.g., rotting plant material, bark, leaves and feces; Whitten et al., 1993). A set of morphological, biochemical and behavioral adaptations thereby enable the location, collection and storage of volatile compounds (Eltz et al., 2005b) forming complex species-specific blends that are stored in tibial organs on the hindlegs and exposed by male euglossine bees during courtship in the forest understory (Eltz et al., 2005a,b). The blends are presumedly used to communicate species affiliation (Eltz et al., 2006; Zimmermann et al., 2006) and/or to demonstrate genetic fitness to conspecific females (Zimmermann et al., 2009b). However, the precise function of perfume blends in mediating mating decision by females awaits experimental support.

The perfume collection behavior of male euglossine bees has the unique feature that the olfactory system is involved in determining both signal production (i.e., the collection of volatile compounds) and signal detection (e.g., during mating) by female bees. Therefore, a strong selection pressure is expected to act on the olfactory system which could lead to sensory specialization in favor of an increased sensitivity to specific single volatiles or volatile blends in different species of euglossine bees (Eltz et al., 2006). In addition to the higher-level neural processing that takes place in the insect brain (see e.g., Renou, 2014), olfactory specialization can be achieved through changes in the peripheral sensory system, for example, by the presence/absence and abundance of specific types of ORs or by divergent chemical tuning of individual ORs (Hallem and Carlson, 2006; Renou, 2014). So far, more than 40 different chemical compounds are known to attract male euglossine bees (Williams and Whitten, 1983; Ramírez et al., 2002; Roubik and Hanson, 2004). Although there is a broad overlap in the range of compounds collected by different species, subgenera or genera of euglossine bees (Ackerman, 1983; Pearson and Dressler, 1985), several studies support a scenario of high species-specific preferences (Ackerman, 1989) as illustrated by the species-specific chemical blends stored in the hind-legs (Eltz et al., 2003, 2005a; Zimmermann et al., 2006; Weber et al., 2016).

This behavior evolved at least 38 million years ago (Engel, 1999; Ramírez et al., 2011) and various neotropical plants, mainly orchids, have adapted to attract male euglossine bees as pollinators by offering volatile compounds as floral reward (i.e., perfume-rewarding plants; Vogel, 1966; Dressler, 1982; Williams and Whitten, 1983; Ramírez et al., 2002). The mutualistic system between euglossine males and perfume-rewarding flowers involves diverse bee genera, which differ considerably in size/morphology, olfactory preferences and behavior (Dressler, 1982; Ramírez et al., 2002). Some of the plants pollinated by male euglossine bees attract many distinct species from all genera, irrespective of their body size (e.g., Anthurium spp. and Spathiphyllum spp.; Montalvo and Ackerman, 1986; Hentrich et al., 2010). However, mutualistic interactions can also be very specific if pollinator size is essential to ensure successful pollinarium removal and subsequent deposition. This is often the case in perfume-producing orchids (e.g., Dodson, 1962, 1978; Dressler, 1968; Meeuse and Morris, 1984).

The orchid genus Catasetum is mainly pollinated by species of Euglossa and Eulaema, but for a few species also pollination by Eufriesea (Hills et al., 1972; Peruquetti et al., 1999; Milet-Pinheiro et al., 2018) and Exaerete (Cancino and Damon, 2007) is reported. Species that are pollinated by Euglossa are usually visited by two or more congeneric pollinator species, but rarely by species of Eulaema, and vice versa (Frankie et al., 1983; Whitten et al., 1986, 1988). Chemical analysis of floral scents emitted by Catasetum orchids suggest that they differ among pollinator genera and subgenera (i.e., Eufriesea, Euglossa or Eulaema; Milet-Pinheiro and Gerlach, 2017; Brandt et al., 2019) but are also highly specific on the species level. Based on these findings, together with the fact that perfume as floral reward has evolved after perfume-gathering behavior, it has been hypothesized that preexisting sensory biases of each euglossine genus and the resulting behavioral preferences for distinct compounds among euglossine bees shaped the evolution of floral scent of perfume-rewarding plants (Ramírez et al., 2011). Experimental evidence for the possible influence of sensory biases on the evolution of floral scents of perfume-rewarding plants from the pollinator perspective, however, is missing. In the present study, we used electroantennography (EAG) to investigate, in a comparative approach, whether bees of the distinct genera Eufriesea, Euglossa, Eulaema, and Exaerete respond differently to chemical compounds that are most representative in the floral perfumes of euglossinophilous plants, particularly in the genus Catasetum (Milet-Pinheiro and Gerlach, 2017). We expect the antennal response profiles of euglossine species to differ among distinct genera thereby reflecting differences in the olfactory periphery of euglossine species that could have influenced the evolution of the floral scents in perfume flowers. Moreover, we conducted a comparative phylogenetic analysis to test whether antennal responses can be explained by bee phylogeny.

In support to our hypothesis, the data revealed that antennal responses differ among euglossine genera, subgenera and species. Antennal responses to the chemical compounds methyl o-anisate, α-copaene, eugenol, β-ionone, ipsdienol, methyl salicylate and (−)-(E)-carvone epoxide were most responsible for these differences. Our phylogenetic analyses revealed that antennal response profiles to some compounds exhibit a phylogenetic signal and the variation in responses across the phylogeny are congruent with a Brownian motion model of trait evolution. This was the case with the antennal responses to benzyl alcohol, 1,4-dimetoxy-benzene, eugenol, linalool, methyl benzoate, methyl salicylate and veratrole. Our data also demonstrates that throughout the evolutionary history of the species we tested, the overall disparity in response between species was greater than expected under a null model of Brownian evolution.

The observed variation among antennal response profiles of tested euglossine bee species and taxonomic groups suggest that the antennae of the different bee species possess distinct types of ORs for different chemical compounds or different amounts of specific ORs. However, previous studies have shown that the sensitivity of ORs can also be influenced by further processes, such as tuning via metabotropic auto-regulation (Getahun et al., 2013) or variability in molecular receptive ranges (Hallem and Carlson, 2006), demonstrating the complexity of the olfactory periphery that could be responsible for the different antennal responses among tested species. Neural processing in the insect brain could also influence the olfactory perception in euglossine bees (e.g., Renou, 2014). To investigate the antennal responses of euglossine bees to specific compounds on the neuronal level, several approaches can be taken, including assaying individual olfactory receptors or measuring neural activity of brain regions in vivo (see e.g., Renou, 2014). For example, methods like single sensillum recording (SSR), the empty neuron system (Brand et al., 2020), or calcium imaging of glomerular responses in the antennal lobe (Galizia and Vetter, 2004) could further contribute to the understanding of the complexity of olfactory tuning, processing and encoding in euglossine bees to chemical compounds that are used during courtship display and that several plants lineages, including orchids, have exploited for pollination services. In addition, sequences of the genome of all tested species could be used in further phylogenetic investigations to study the diversity of OR genes.

The results of our electroantennographic analyses revealed a clear difference among the antennal response profiles among euglossine bee genera (especially between Euglossa and Eulaema). Our study offers the first experimental evidence for the assumption that properties of the sensory equipment assort according to major taxonomic groups of euglossine bees. Bees of different genera respond differently to compounds, such as α-copaene, eugenol, ipsdienol, and (−)-(E)-carvone epoxide. In agreement to these patterns, the chemical composition of floral scents of perfume-rewarding orchids has been shown to differ among Euglossa- and Eulaema-pollinated species. Several chemical compounds which seem to be typically found in the floral scent bouquets of either Euglossa- (i.e., ipsdienol and myrcene; Milet-Pinheiro and Gerlach, 2017; Brandt et al., 2020) or Eulaema-pollinated species (e.g., α-pinene and (−)-(E)-carvone epoxide; Whitten et al., 1986; Milet-Pinheiro and Gerlach, 2017) coincide with the compounds most responsible for the separation of antennal response profiles among tested euglossine genera in our study. Altogether, these findings underline the finding of Ramírez et al. (2011) suggesting that distinct sensory biases between euglossine bee lineages have shaped the evolution of floral scents in perfume-rewarding plants. Under such scenario, floral scent bouquets evolve to target the compounds with strong sensory responses and behavioral attraction (Milet-Pinheiro and Gerlach, 2017) and lead to a genus specific attraction of pollinators. This is important because of the highly specialized pollination mechanisms exhibited by some perfume-producing orchids (see Dodson, 1962; Vogel, 1966; Romero and Carnevali, 2009) in which the morphological properties (i.e., the size) of euglossine bees, which typically differ among genera, ensure successful pollinarium transfer from male to female flowers (Dodson, 1962, 1978). For example, Catasetum species that are pollinated by Euglossa bees (8–18 mm in size; Dressler, 1982; Carvalho and Machado, 2002; Ramírez et al., 2002) are usually visited by two or more congeneric species, but rarely by species of Eulaema with a larger body size (20–35 mm in size; Dressler, 1982; Ramírez et al., 2002), and vice versa (Whitten et al., 1986, 1988; Ramírez et al., 2002). In fact, a similar pattern has also been reported in the orchid genus Gongora, which is also exclusively pollinated by euglossine bees (Hetherington-Rauth and Ramírez, 2015).

Within the tested euglossine bee genera, we found that the antennal response profiles are also specific at the subgenera and species levels. This observation provides further evidence for the idea of sensory niche partitioning provided by Zimmermann et al. (2009a). In areas where many different euglossine species of the same genus occur sympatrically, species-specific attraction of pollinators to perfume-rewarding flowers is not only essential to ensure pollinator fidelity and avoid pollinator competition but also to ensure reproductive isolation among closely related orchid species. Appropriate mixtures of chemical compounds or the presence of specific major compounds in the floral scents enable the attraction of only few out of many different euglossine species (Dodson, 1970; Whitten et al., 1986). Together with further isolating mechanisms (see e.g., Hills et al., 1972; Williams and Whitten, 1983) the resulting highly specific attraction of pollinators in euglossinophilous plants can act as an effective reproductive barrier among otherwise interfertile plant species (Milet-Pinheiro and Gerlach, 2017). This is possible due to compound-specific differences in antennal perception even among closely related euglossine species, as we report here. For example, Eltz et al. (2008) showed how males of Eg. dilemma are strongly attracted to hydroxy-6-nona-1,3-dienyl-benzaldehyde (HNDB) and show strong antennal responses, while bees of the closely related and morphologically (Eltz et al., 2011) as well as ecologically (Villanueva-Gutierrez et al., 2013) similar species Eg. viridissima neither responds to this compound behaviorally nor electroantennographically. Brand et al. (2015, 2020) found that this divergence can be explained by a different selection among one single olfactory receptor gene (i.e., OR41), proving that (1) changes in the chemosensory gene family occur among closely related species and that (2) strong divergent selection acting on chemosensory receptor genes plays an important role in the evolution and diversification of the olfactory system in euglossine bees. The high species-specificity in antennal response profiles among species could be explained by the patterns of evolution of chemical sexual signaling. For example, the study of Cardé and Baker (1984) suggests that female preferences for a signal (receiver) impose strong stabilizing selection on male signal traits (sender), favoring the stability of the signal among populations and leading to a high species-specificity of chemical traits even across large geographic distances (Ord and Stamps, 2009). In this context, Zimmermann et al. (2006) and Ramírez et al. (2010a) revealed a qualitative consistency in perfume composition of tibial organs within euglossine bee species even when comparing populations from distant geographic regions that harbor different perfume sources.

The results of the Blomberg’s K test indicate the presence of a phylogenetic signal in the antennal response profiles of euglossine bee species. More specifically, there seems to be a tendency for species within a lineage to resemble each other more in their antennal responses than they resemble other lineages or random pairs of species, indicating that the diversification of the olfactory system of euglossine bee clades (genera) is phylogenetically conserved. Some chemical compounds (i.e., benzyl alcohol, 1,4-dimetoxy benzene, eugenol, linalool, methyl benzoate, methyl salicylate and veratrole) revealed an optimal level of antennal responses in the Brownian motion or Orenstein-Uhlenbeck model. A similar pattern was already described by Mitko et al. (2016) comparing the antennal responses of males belonging to 15 sympatric Euglossa species stimulated with compounds present in the hind tibiae. The results of this study suggest that sensory specialization has occurred within multiple lineages due to strong antennal responses for some chemicals that are present as major compounds in the perfume of the same species. Such a pattern is congruent with strong stabilizing selection acting to maintain antennal responses to specific compounds across the phylogeny (Hansen, 1997). The compounds affected by that pattern in our study have been frequently reported, not only within the floral scents of perfume-rewarding pollination systems (e.g., Montalvo and Ackerman, 1986; Gerlach and Schill, 1991; Hentrich et al., 2010), but in a variety of angiosperms worldwide (Knudsen et al., 2006). Therefore, we can assume that the selection on antennal response profiles of euglossine bee species could not only be driven by the association of perfume-rewarding plants but also by other aspects. For example, the compounds promoting an optimal level of antennal responses, as suggested by our phylogenetic analysis, could be important signal traits in the discrimination of sex partners by female orchid bees (see also Cardé and Baker, 1984) or in the search for nectar (see e.g., Borrell, 2005). Since perfume-rewarding flowers seem to contribute only little to the aromatic richness found in the tibial organs of male euglossine bees (Whitten et al., 1993; Ramírez et al., 2010a), we also cannot exclude, for example, the influence of non-floral perfume sources for male euglossine bees, such as rotting plant material, bark, leaves, and feces (Whitten et al., 1993).

At the same time, the results derived from the DDT plots indicate that closely related euglossine diverge more quickly on their antennal response profiles than expected under a Brownian motion model of neutral trait evolution, especially at the species level underlining the specificity of antennal responses on the species level of euglossine bees found in our electroantennographic analyses. Generally, these results resemble the patterns that have already been described on the macroevolution of perfume signaling in euglossine bees (i.e., perfumes collected in the tibial organs of males). For example, the study of Weber et al. (2016) revealed both high species-specificity and elevated rates of evolution in perfume signals found in extracts of the tibial organs of distinct Euglossa species and stated that perfume evolution may be tied to the high number of orchid bee species coexisting together in neotropical communities. Furthermore, they described a rapid divergence at speciation and character displacement (see also Zimmermann et al., 2009b). Because of the high diversity of antennal response profiles on the species level, we cannot totally exclude the possibility that there might exist co-evolutionary adaptations between perfume flowers and euglossine bees, especially during the latest stage of evolution. Earlier studies have, indeed, suspected a (rather loose) coevolution for perfume rewarding orchids and their euglossine pollinators (e.g., Kiester et al., 1984). However, the already mentioned findings that (1) floral scent of perfume flowers developed much later than the collection behavior in euglossine bees (Ramírez et al., 2011) and that (2) a great part of chemical compounds collected by euglossine bees to build their unique tibial blends derive from non-floral rather than floral sources (Whitten et al., 1993; Ramírez et al., 2010a), suggest that sexual selection/changes in mating ecology might influence the evolution of the olfactory equipment of distinct euglossine bee species rather than the floral sources or their availability.

Altogether, the results of our study offer an overview of antennal responses for many different euglossine bee species belonging to distinct genera. The differences in antennal responses between distinct euglossine genera and subgenera, as well as species-specific patterns, reinforce the findings for the floral scent compositions in different species of perfume-rewarding flowers and offer first experimental evidence for the hypothesis of pollinator-mediated selection of floral scents driven by preexisting sensory biases in euglossine bees (Ramírez et al., 2011). The findings of our phylogenetic analyses indicate that a diversification of the olfactory system between euglossine bee genera could be (at least partly) phylogenetically conserved. Moreover, our results are congruent with a scenario of stabilizing selection acting on antennal responses to individual compounds, in particular to chemical compounds commonly found in perfume-rewarding flowers. At the same time, closely related species within taxonomic groups can differ considerably in their olfactory system due to a rapid evolution and a high level of disparity (Brand et al., 2020). Further phylogenetic investigations, for example on chemosensory genes of euglossine species (similar to the work of Brand et al., 2015) in combination with electroantennographic comparisons could shed more light into the evolution of the sensory periphery of euglossine bees and, consequently, in the evolution of floral scents in perfume-rewarding flowers.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

KB, SD, PM-P, and MA developed the experimental design and the idea. OR and DD synthetized the tested compound carvone epoxide. KB and PM-P conducted all experiments and collected the data. KB wrote the first draft of the manuscript and analyzed the results of the electroantennography. SR was mainly responsible for the phylogenetic analyses. All authors contributed to the revision of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.