Santiago R. Ramírez

Dating the origin of the Orchidaceae from a fossil orchid with its pollinator

Since the time of Darwin, evolutionary biologists have been fascinated by the spectacular adaptations to insect pollination exhibited by orchids. However, despite being the most diverse plant family on Earth, the Orchidaceae lack a definitive fossil record and thus many aspects of their evolutionary history remain obscure. Here we report an exquisitely preserved orchid pollinarium (of Meliorchis caribea gen. et sp. nov.) attached to the mesoscutellum of an extinct stingless bee, Proplebeia dominicana, recovered from Miocene amber in the Dominican Republic, that is 15–20 million years (Myr) old. This discovery constitutes both the first unambiguous fossil of Orchidaceae and an unprecedented direct fossil observation of a plant–pollinator interaction. By applying cladistic methods to a morphological character matrix, we resolve the phylogenetic position of M. caribea within the extant subtribe Goodyerinae (subfamily Orchidoideae). We use the ages of other fossil monocots and M. caribea to calibrate a molecular phylogenetic tree of the Orchidaceae. Our results indicate that the most recent common ancestor of extant orchids lived in the Late Cretaceous (76–84 Myr ago), and also suggest that the dramatic radiation of orchids began shortly after the mass extinctions at the K/T boundary. These results further support the hypothesis of an ancient origin for Orchidaceae.

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.

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.

Deciphering the language of plant communication: volatile chemotypes of sagebrush

Volatile communication between sagebrush (Artemisia tridentata) individuals has been found previously to reduce herbivory and to be more effective between individuals that are genetically identical or related relative to between strangers. The chemical nature of the cues involved in volatile communication remains unknown for this and other systems. We collected headspace volatiles from sagebrush plants in the field and analyzed these using GC-MS. Volatile profiles were highly variable among individuals, but most individuals could be characterized as belonging to one of two chemotypes, dominated by either thujone or camphor. Analyses of parents and offspring revealed that chemotypes were highly heritable. The ecological significance of chemotypes and the genetic mechanisms that control them remain poorly understood. However, we found that individuals of the same chemotype communicated more effectively and experienced less herbivory than individuals of differing chemotypes. Plants may use chemotypes to distinguish relatives from strangers.

A molecular phylogeny of the stingless bee genus Melipona (Hymenoptera: Apidae)

Stingless bees (Meliponini) constitute a diverse group of highly eusocial insects that occur throughout tropical regions around the world. The meliponine genus Melipona is restricted to the New World tropics and has over 50 described species. Melipona, like Apis, possesses the remarkable ability to use representational communication to indicate the location of foraging patches. Although Melipona has been the subject of numerous behavioral, ecological, and genetic studies, the evolutionary history of this genus remains largely unexplored. Here, we implement a multigene phylogenetic approach based on nuclear, mitochondrial, and ribosomal loci, coupled with molecular clock methods, to elucidate the phylogenetic relationships and antiquity of subgenera and species of Melipona. Our phylogenetic analysis resolves the relationship among subgenera and tends to agree with morphology-based classification hypotheses. Our molecular clock analysis indicates that the genus Melipona shared a most recent common ancestor at least approximately 14-17 million years (My) ago. These results provide the groundwork for future comparative analyses aimed at understanding the evolution of complex communication mechanisms in eusocial Apidae.

Diversification and phylogeographic structure in widespread Azteca plant‐ants from the northern Neotropics

The Neotropical myrmecophytic tree Cordia alliodora hosts symbiotic Azteca ants in most of its widespread range. The taxonomy of the genus Azteca is notoriously difficult, which has frequently obscured species identity in ecological studies. We used sequence data from one mitochondrial and four nuclear loci to infer phylogenetic relationships, patterns of geographic distribution, and timing of diversification for 182 colonies of five C. alliodora‐dwelling Azteca species from Mexico to Colombia. All morphological species were recovered as monophyletic, but we identified at least five distinct genetic lineages within the most abundant and specialized species, Azteca pittieri. Mitochondrial and nuclear data were concordant at the species level, but not within species. Divergence time analyses estimated that C. alliodora‐dwelling Azteca shared a common ancestor approximately 10–22 million years ago, prior to the proposed arrival of the host tree in Middle America. Diversification in A. pittieri occurred in the Pleistocene and was not correlated with geographic distance, which suggests limited historical gene flow among geographically restricted populations. This contrasts with the previously reported lack of phylogeographic structure at this spatial scale in the host tree. Climatic niches, and particularly precipitation‐related variables, did not overlap between the sites occupied by northern and southern lineages of A. pittieri. Together, these results suggest that restricted gene flow among ant populations may facilitate local adaptation to environmental heterogeneity. Differences in population structure between the ants and their host trees may profoundly affect the evolutionary dynamics of this widespread ant–plant mutualism.

Intraspecific Geographic Variation of Fragrances Acquired by Orchid Bees in Native and Introduced Populations

Male orchid bees collect volatiles, from both floral and non-floral sources, that they expose as pheromone analogues (perfumes) during courtship display. The chemical profile of these perfumes, which includes terpenes and aromatic compounds, is both species-specific and divergent among closely related lineages. Thus, fragrance composition is thought to play an important role in prezygotic reproductive isolation in euglossine bees. However, because orchid bees acquire fragrances entirely from exogenous sources, the chemical composition of male perfumes is prone to variation due to environmental heterogeneity across habitats. We used Gas Chromatography/Mass Spectrometry (GC/MS) to characterize the perfumes of 114 individuals of the green orchid bee (Euglossa aff. viridissima) sampled from five native populations in Mesoamerica and two naturalized populations in the southeastern United States. We recorded a total of 292 fragrance compounds from hind-leg extracts, and found that overall perfume composition was different for each population. We detected a pronounced chemical dissimilarity between native (Mesoamerica) and naturalized (U.S.) populations that was driven both by proportional differences of common compounds as well as the presence of a few chemicals unique to each population group. Despite these differences, our data also revealed remarkable qualitative consistency in the presence of several major fragrance compounds across distant populations from dissimilar habitats. In addition, we demonstrate that naturalized bees are attracted to and collect large quantities of triclopyr 2-butoxyethyl ester, the active ingredient of several commercially available herbicides. By comparing incidence values and consistency indices across populations, we identify putative functional compounds that may play an important role in courtship signaling in this species of orchid bee.

Variation in the ability to communicate three-dimensional resource location by stingless bees from different habitats

We evaluated the ability of two Brazilian stingless bee species, Melipona mandacaia and M. bicolor ,t o recruit nestmates to a specific three-dimensional location. We used experimental feeder arrays and provide the first detailed evidence demonstrating that recruitment communication in Melipona can lead to large, rapid and highly significant increases in the number of nestmates visiting a specific location. Melipona bicolor and M. mandacaia foragers both recruited nestmates to the correct distance and direction, but differed in their ability to recruit nestmates to the correct height. These differences may relate to their respective habitats. Melipona mandacaia inhabits semi-arid areas of Caatinga where most food sources occur close to the ground, and its foragers evidently cannot recruit nestmates to the correct height. Melipona bicolor, an Atlantic rainforest species, evidently does not communicate height when the food source is at ground level, but can communicate height when the food source is at the forest canopy level (12 m high), where major food sources occur. Species-specific variation in three-dimensional location communication is intriguing because it suggests that Melipona may be a good model for studying the evolution of recruitment communication systems in highly social bees.  2003 Published by Elsevier Ltd on behalf of The Association for the Study of Animal Behaviour.

Rapid evolution of chemosensory receptor genes in a pair of sibling species of orchid bees (Apidae: Euglossini)

BackgroundInsects rely more on chemical signals (semiochemicals) than on any other sensory modality to find, identify, and choose mates. In most insects, pheromone production is typically regulated through biosynthetic pathways, whereas pheromone sensory detection is controlled by the olfactory system. Orchid bees are exceptional in that their semiochemicals are not produced metabolically, but instead male bees collect odoriferous compounds (perfumes) from the environment and store them in specialized hind-leg pockets to subsequently expose during courtship display. Thus, the olfactory sensory system of orchid bees simultaneously controls male perfume traits (sender components) and female preferences (receiver components). This functional linkage increases the opportunities for parallel evolution of male traits and female preferences, particularly in response to genetic changes of chemosensory detection (e.g. Odorant Receptor genes). To identify whether shifts in pheromone composition among related lineages of orchid bees are associated with divergence in chemosensory genes of the olfactory periphery, we searched for patterns of divergent selection across the antennal transcriptomes of two recently diverged sibling species Euglossa dilemma and E. viridissima.ResultsWe identified 3185 orthologous genes including 94 chemosensory loci from five different gene families (Odorant Receptors, Ionotropic Receptors, Gustatory Receptors, Odorant Binding Proteins, and Chemosensory Proteins). Our results revealed that orthologs with signatures of divergent selection between E. dilemma and E. viridissima were significantly enriched for chemosensory genes. Notably, elevated signals of divergent selection were almost exclusively observed among chemosensory receptors (i.e. Odorant Receptors).ConclusionsOur results suggest that rapid changes in the chemosensory gene family occurred among closely related species of orchid bees. These findings are consistent with the hypothesis that strong divergent selection acting on chemosensory receptor genes plays an important role in the evolution and diversification of insect pheromone systems.

Evolution and diversity of floral scent chemistry in the euglossine bee-pollinated orchid genus Gongora

•Background and Aims Animal-pollinated angiosperms have evolved a variety of signalling mechanisms to attract pollinators. Floral scent is a key component of pollinator attraction, and its chemistry modulates both pollinator behaviour and the formation of plant–pollinator networks. The neotropical orchid genus Gongora exhibits specialized pollinator associations with male orchid bees (Euglossini). Male bees visit orchid flowers to collect volatile chemical compounds that they store in hind-leg pouches to use subsequently during courtship display. Hence, Gongora floral scent compounds simultaneously serve as signalling molecules and pollinator rewards. Furthermore, because floral scent acts as the predominant reproductive isolating barrier among lineages, it has been hypothesized that chemical traits are highly species specific. A comparative analysis of intra- and inter-specific variation of floral scent chemistry was conducted to investigate the evolutionary patterns across the genus. •Methods Gas chromatography–mass spectrometry (GC-MS) was used to analyse the floral scent of 78 individuals belonging to 28 different species of Gongora from two of the three major lineages sampled across the neotropical region. Multidimensional scaling and indicator value analyses were implemented to investigate the patterns of chemical diversity within and among taxonomic groups at various geographic scales. Additionally, pollinator observations were conducted on a sympatric community of Gongora orchids exhibiting distinct floral scent phenotypes. •Key Results A total of 83 floral volatiles, mainly terpenes and aromatic compounds, were detected. Many of the identified compounds are common across diverse angiosperm families (e.g. cineole, eugenol, β-ocimene, β-pinene and terpinen-4-ol), while others are relatively rare outside euglossine bee-pollinated orchid lineages. Additionally, 29 volatiles were identified that are known to attract and elicit collection behaviour in male bees. Floral scent traits were less variable within species than between species, and the analysis revealed exceptional levels of cryptic diversity. Gongora species were divided into 15 fragrance groups based on shared compounds. Fragrance groups indicate that floral scent variation is not predicted by taxonomic rank or biogeographic region. •Conclusions Gongora orchids emit a diverse array of scent molecules that are largely species specific, and closely related taxa exhibit qualitatively and quantitatively divergent chemical profiles. It is shown that within a community, Gongora scent chemotypes are correlated with near non-overlapping bee pollinator assemblies. The results lend support to the hypothesis that floral scent traits regulate the architecture of bee pollinator associations. Thus, Gongora provides unique opportunities to examine the interplay between floral traits and pollinator specialization in plant–pollinator mutualisms.