Thomas Eltz

FRAGRANCE COLLECTION, STORAGE, AND ACCUMULATION BY INDIVIDUAL MALE ORCHID BEES

Individually marked males of two species of Euglossa were sighted repeatedly and over considerable periods of time (up to 44 days) at artificial fragrance baits exposed on Barro Colorado Island (BCI), Panama. Individuals switched between different bait chemicals that are attractive for the respective species, and no bait preferences or individual bait constancy was observed. GC-MS analyses of 153 males of three species showed that individual hind tibiae contain highly variable quantities of a complex and species-specific blend of fragrance compounds, mainly terpenoids and aromatics. In all three species, frequency distributions of individual quantities were strongly skewed towards individuals with small amounts, and individual amount and complexity were positively correlated. Tibial contents of male Euglossa imperialis that were kept alive in a flight cage for 0, 5, 10, or 15 days showed no qualitative or quantitative change over time, suggesting that the fragrances are very efficiently stored in the hind legs. In Euglossa cognata wing wear, an established age correlate of the species, was positively correlated with individual fragrance quantity. Our results suggest that male euglossines forage continuously for a variety of volatiles, store them, and finally acquire large quantities of a complex and specific fragrance bouquet. Both qualitative and quantitative aspects of individual contents are likely to contain information on male phenotypic and genotypic quality.

Size does matter - effects of tropical rainforest fragmentation on the leaf litter ant community in Sabah, Malaysia

Primary tropical lowland rainforest in Sabah, Malaysia, has been largely reduced to small to medium-sized, often isolated, forest islands surrounded by a highly altered agricultural landscape. The biodiversity patterns of leaf litter ant communities were monitored in two forest fragments of differing size as well as in a contiguous forest over the course of two years. Species number and diversity in the forest isolates was significantly lower, reaching only 47.5% of the species number collected in the contiguous forest. Species density was also lower, which had led to a thinning of the ant community in the fragments. Community composition was substantially altered in the forest remnants, and an increase of tramp species with smaller fragment size was detected. These results were unexpected and alarming, as the medium-sized forest is with its 42.9 km2 a comparatively large primary forest fragment for Sabah.

Juggling with volatiles: exposure of perfumes by displaying male orchid bees

Male orchid bees (Euglossini) pollinate 10% of the neotropical orchid flora while collecting floral scents, which they store and accumulate in hind tibial pouches. The purpose of these fragrances is unclear, as is the context, timing and mechanism of their possible exposure. Here we show for the first time that males expose and relocate their fragrances during courtship display. We present high-speed video analyses revealing an intricate and repetitive leg movement performed by displaying male Euglossa cognata. The behavior involves several morphological structures of hitherto unknown function and suggests transfer of substances from the hind tibia to a contralateral mid-tibial tuft of hairs. Body-side-specific fluorescent dye application and consecutive detection of signals on males after display confirmed this transfer. Deposited on the mid-tibial tufts, the fragrances are ideally placed in order to become ventilated by jugal combs on the wing bases, as previously suggested by Bembé (in Apidologie 35:288–291, 2004). Being clearly distinct from motor patterns involved in fragrance collection, the described movement is continuously performed by displaying males, suggesting an equally continuous exposure of volatiles. Although the findings strengthen, the view that the volatiles serve as attractants in the context of mating behavior, the signal addressee, conspecific males or females, has yet to be found.

Asynchronous Diversification in a Specialized Plant-Pollinator Mutualism

Insect sensory biases played a major role in driving reproductive adaptations in flowering plants. Most flowering plants establish mutualistic associations with insect pollinators to facilitate sexual reproduction. However, the evolutionary processes that gave rise to these associations remain poorly understood. We reconstructed the times of divergence, diversification patterns, and interaction networks of a diverse group of specialized orchids and their bee pollinators. In contrast to a scenario of coevolution by race formation, we show that fragrance-producing orchids originated at least three times independently after their fragrance-collecting bee mutualists. Whereas orchid diversification has apparently tracked the diversification of orchids’ bee pollinators, bees appear to have depended on the diverse chemical environment of neotropical forests. We corroborated this apparent asymmetrical dependency by simulating co-extinction cascades in real interaction networks that lacked reciprocal specialization. These results suggest that the diversification of insect-pollinated angiosperms may have been facilitated by the exploitation of preexisting sensory biases of insect pollinators.

Foraging scent marks of bumblebees: footprint cues rather than pheromone signals.

In their natural habitat foraging bumblebees refuse to land on and probe flowers that have been recently visited (and depleted) by themselves, conspecifics or other bees, which increases their overall rate of nectar intake. This avoidance is often based on recognition of scent marks deposited by previous visitors. While the term ‘scent mark’ implies active labelling, it is an open question whether the repellent chemicals are pheromones actively and specifically released during flower visits, or mere footprints deposited unspecifically wherever bees walk. To distinguish between the two possibilities, we presented worker bumblebees (Bombus terrestris) with three types of feeders in a laboratory experiment: unvisited control feeders, passive feeders with a corolla that the bee had walked over on its way from the nest (with unspecific footprints), and active feeders, which the bee had just visited and depleted, but which were immediately refilled with sugar–water (potentially with specific scent marks). Bumblebees rejected both active and passive feeders more frequently than unvisited controls. The rate of rejection of passive feeders was only slightly lower than that of active feeders, and this difference vanished completely when passive corollas were walked over repeatedly on the way from the nest. Thus, mere footprints were sufficient to emulate the repellent effect of an actual feeder visit. In confirmation, glass slides on which bumblebees had walked on near the nest entrance accumulated hydrocarbons (alkanes and alkenes, C23 to C31), which had previously been shown to elicit repellency in flower choice experiments. We conclude that repellent scent marks are mere footprints, which foraging bees avoid when they encounter them in a foraging context.

Chemical niche differentiation among sympatric species of orchid bees

Male Neotropical orchid bees (Euglossini) collect volatile substances (fragrances) from flowers and other sources (e.g., decaying wood) and store them in specialized hind tibial pockets. The accumulated chemicals are later emitted during courtship display, presumably to lure conspecific females for mating. We analyzed tibial fragrances of males of 15 sympatric Panamanian species in the genus Euglossa to test whether communities of euglossine bees are chemically structured, and to elucidate whether male fragrance signals evolve to convey premating isolation. Our analysis revealed substantial chemical disparity among all lineages. Disparity was mediated by compounds that were exclusive to certain species but also by differences in relative quantity of shared compounds. We mapped tibial fragrance compounds present in each species on a DNA-based phylogeny (reconstructed using partial sequences of COI, EF1-alpha, ArgK, and Pol-II) and found that most dominant compounds were highly homoplasious. In an analysis of chemical differentiation in relation to phylogenetic divergence through time, disparity was greater than expected from a null model at any point during evolutionary history, suggesting that diversifying selection has shaped fragrance phenotypes. Notably, chemical disparity was greater within recently diverged lineages than among them, suggesting that chemical preferences in orchid bees evolved rapidly in the early stages of species divergence. We postulate communication interference as the possible mechanism behind the observed fragrance differentiation, which may be the product of reproductive character (fragrance) displacement. Our findings are consistent with the hypothesis that male fragrance signals evolve to convey premating isolation.

Pollen foraging and resource partitioning of stingless bees in relation to flowering dynamics in a Southeast Asian tropical rainforest

Summary: We used microscopic pollen analysis to investigate the diversity and similarity of pollen diets of six colonies of stingless bees (Apidae; Meliponini) located within one monospecific (three colonies of Trigona collina) and one mixed nesting aggregation (one colony of T. collina, and one colony of each of the close relatives T. melina and T. melanocephala) in lowland tropical rain forest in Sabah, Malaysia. Samples of 20 corbicular loads, collected six times over a period of three months from each colony, contained a total of 74 different morphotypes of pollen grains with an average between 4.7 to 8.5 per sample for the different colonies. In an analysis on total diet composition intraspecific similarity was much greater than interspecific similarity. The focal colony of Trigona collina from the mixed aggregation distinctly clustered according to species rather than nest location, suggesting that some interspecific resource partitioning occurs. The sampling period was accompanied by a drastic increase in flowering activity as evidenced by data from a flower phenology transect. At times of limited flowering similarity of pollen diets was generally low, both within and between species. It is hypothesized that this is so because bees are forced to forage from scattered subsets of flower patches spread out over a large foraging range. In times of increased flowering pollen diet similarity significantly increased between colonies of the monospecific aggregation, presumably because colonies concentrated on more profitable sources in closer proximity. In contrast, similarity remained low within the mixed aggregation, suggesting that innate differences in foraging preferences precluded any effect of diet convergence.

Species-specific attraction to pheromonal analogues in orchid bees

Male orchid bees (Euglossini) collect fragrances from flowers and other natural sources, a behaviour that has shaped the euglossine pollination syndrome. Males store such chemicals in hind leg pouches and later expose them during courtship display. In the present study, we show that complex bouquets of two sympatric species of Eulaema, E. meriana and E. bombiformis, are chemically distinct. When exposed during bioassays at display perches individual hind leg extracts rapidly and consistently attracted other males of the correct species, even if derived from males of disparate localities (French Guiana and Panama). Conspecific males as well as females of E. bombiformis arrived at natural perch sites only from downwind, and two copulations were observed. Our findings demonstrate that acquired odours mediate exclusive attraction within species and support the idea that such fragrances are pheromone analogues. Their role in acquiring matings and during male–male interaction is discussed.

An olfactory shift is associated with male perfume differentiation and species divergence in orchid bees.

Saltational changes may underlie the diversification of pheromone communication systems in insects, which are normally under stabilizing selection favoring high specificity in signals and signal perception. In orchid bees (Euglossini), the production of male signals depends on the sense of smell: males collect complex blends of volatiles (perfumes) from their environment, which are later emitted as pheromone analogs at mating sites. We analyzed the behavioral and antennal response to perfume components in two male morphotypes of Euglossa cf. viridissima from Mexico, which differ in the number of mandibular teeth. Tridentate males collected 2-hydroxy-6-nona-1,3-dienyl-benzaldehyde (HNDB) as the dominant component of their perfume. In bidentate males, blends were broadly similar but lacked HNDB. Population genetic analysis revealed that tri- and bidentate males belong to two reproductively isolated lineages. Electroantennogram tests (EAG and GC-EAD) showed substantially lower antennal responses to HNDB in bidentate versus tridentate males, revealing for the first time a mechanism by which closely related species acquire different chemical compounds from their habitat. The component-specific differences in perfume perception and collection in males of two sibling species are in agreement with a saltational, olfaction-driven mode of signal perfume evolution. However, the response of females to the diverged signals remains unknown.

Enfleurage, lipid recycling and the origin of perfume collection in orchid bees

Enfleurage, the extraction of elusive floral scents with the help of a lipophilic carrier (grease), is widely used in the perfume industry. Male neotropical orchid bees (Euglossini), which accumulate exogenous fragrances as pheromone analogues, use a similar technique. To collect fragrances, the bees apply large amounts of straight-chain lipids to odoriferous surfaces from their cephalic labial glands, which dissolve the volatiles, and the mixture is then transferred to voluminous hind-leg pockets. Here, we show that males do in fact operate a lipid conveyor belt to accumulate and concentrate their perfume. From the hind-leg pockets of caged male Euglossa viridissima, deuterated derivatives of carrier lipids were consecutively sequestered, shuttled back to the labial glands and reused on consecutive bouts of fragrance collection. Such lipid cycling is instrumental in creating complex perfume bouquets. Furthermore, we found that labial glands of male orchid bees are strikingly similar to those of scent-marking male bumblebees in terms of size, form and structure. This, and a prominent overlap in secretory products, led us to propose that perfume collection evolved from scent-marking in ancestral corbiculate bees.