Norris H. Williams

ORCHID FLORAL FRAGRANCES AND MALE EUGLOSSINE BEES: METHODS AND ADVANCES IN THE LAST SESQUIDECADE

All species of the Neotropical subtribes Stanhopeinae and Catasetinae (Orchi daceae) are pollinated exclusively by male euglossine bees which are attracted to and collect the floral fragrances. The orchid-euglossine bee relationship is often highly specific:the flower of a given species of plant may attract males of only one or a few species out of dozens of euglossine species in the habitat. This pollinator specificityis based upon species-specificcombinations of floralfragrancecompounds which attractonly one or a few species of euglossine bees. Such pollinator specificity is an important reproductive isolating mechanism between sympatric interfertile species of orchids. The male bees are thought to use the collected floral fragrance compounds in their own reproductivebiology, probably as precursorsof their own sex pheromones.

Subtribal and generic relationships of Maxillarieae (Orchidaceae) with emphasis on Stanhopeinae: combined molecular evidence.

The monophyly of and phylogenetic relationships within the orchid tribe Maxillarieae Pfitzer were evaluated using parsimony analyses of combined nuclear ribosomal and plastid DNA sequence data of ITS 1 and 2, matK, and the trnL intron and the trnL-F intergene spacer. Each of the separate analyses produced highly congruent but weakly supported patterns (by the bootstrap), so these were combined in a single analysis. Analysis of 90 ingroup taxa (representing ∼35% of currently recognized genera) and four outgroup taxa produced resolved and highly supported cladograms. Based on the cladograms, we recognize six subtribes: Eriopsidinae, Oncidiinae (including Pachyphyllinae, Ornithocephalinae, and Telipogoninae), Stanhopeinae, Coeliopsidinae, Maxillariinae (including Lycastinae and Bifrenariinae), and Zygopetalinae (including Cryptarrheninae, Dichaeinae, Huntleyinae, and Warreinae). Stanhopeinae were sampled most intensively; their generic relationships were highly resolved in the analysis and largely agree with currently accepted generic concepts based on morphology. Coeliopsidinae (Coeliopsis, Lycomormium, Peristeria) are sister to Stanhopeinae. Correlations are drawn among phylogeny, pollination mechanisms, and life history traits.

Orchid phylogenomics and multiple drivers of their extraordinary diversification

Orchids are the most diverse family of angiosperms, with over 25 000 species, more than mammals, birds and reptiles combined. Tests of hypotheses to account for such diversity have been stymied by the lack of a fully resolved broad-scale phylogeny. Here, we provide such a phylogeny, based on 75 chloroplast genes for 39 species representing all orchid subfamilies and 16 of 17 tribes, time-calibrated against 17 angiosperm fossils. A supermatrix analysis places an additional 144 species based on three plastid genes. Orchids appear to have arisen roughly 112 million years ago (Mya); the subfamilies Orchidoideae and Epidendroideae diverged from each other at the end of the Cretaceous; and the eight tribes and three previously unplaced subtribes of the upper epidendroids diverged rapidly from each other between 37.9 and 30.8 Mya. Orchids appear to have undergone one significant acceleration of net species diversification in the orchidoids, and two accelerations and one deceleration in the upper epidendroids. Consistent with theory, such accelerations were correlated with the evolution of pollinia, the epiphytic habit, CAM photosynthesis, tropical distribution (especially in extensive cordilleras), and pollination via Lepidoptera or euglossine bees. Deceit pollination appears to have elevated the number of orchid species by one-half but not via acceleration of the rate of net diversification. The highest rate of net species diversification within the orchids (0.382 sp sp−1 My−1) is 6.8 times that at the Asparagales crown.

Evolution along the crassulacean acid metabolism continuum

Crassulaceanacid metabolism(CAM) isaspecialised modeof photosynthesisthat improves atmospheric CO2 assimilationinwater-limited terrestrial andepiphytichabitatsandinCO2-limited aquatic environments.Incontrast withC3 and C4 plants, CAM plants take up CO2 from the atmosphere partially or predominantly at night. CAM is taxonomically widespreadamongvascularplantsandispresentinmanysucculentspeciesthatoccupysemiaridregions,aswellasintropical epiphytesandinsomeaquaticmacrophytes.Thiswater-conservingphotosyntheticpathwayhasevolvedmultipletimesand isfoundincloseto6%ofvascularplantspeciesfromatleast35families.AlthoughmanyaspectsofCAMmolecularbiology, biochemistryandecophysiologyarewellunderstood,relativelylittleisknownabouttheevolutionaryoriginsofCAM.This review focuses on five main topics: (1) the permutations and plasticity of CAM, (2) the requirements for CAM evolution, (3) the drivers of CAM evolution, (4) the prevalence and taxonomic distribution of CAM among vascular plants with emphasisontheOrchidaceaeand(5)themolecularunderpinningsofCAMevolutionincludingcircadianclockregulationof gene expression.

Phylogenetic utility of ycf1 in orchids: a plastid gene more variable than matK

Plastid DNA sequences have been widely used by systematists for reconstructing plant phylogenies. The utility of any DNA region for phylogenetic analysis is determined by ease of amplification and sequencing, confidence of assessment in phylogenetic character alignment, and by variability across broad taxon sampling. Often, a compromise must be made between using relatively highly conserved coding regions or highly variable introns and intergenic spacers. Analyses of a combination of these types of DNA regions yield phylogenetic structure at various levels of a tree (i.e., along the spine and at the tips of the branches). Here, we demonstrate the phylogenetic utility of a heretofore unused portion of a plastid protein-coding gene, hypothetical chloroplast open reading frame 1 (ycf1), in orchids. All portions of ycf1 examined are highly variable, yet alignable across Orchidaceae, and are phylogenetically informative at the level of species. In Orchidaceae, ycf1 is more variable than matK both in total number of parsimony informative characters and in percent variability. The nrITS region is more variable than ycf1, but is more difficult to align. Although we only demonstrate the phylogenetic utility of ycf1 in orchids, it is likely to be similarly useful among other plant taxa.

Molecular phylogenetics of Vandeae (Orchidaceae) and the evolution of leaflessness

Members of tribe Vandeae (Orchidaceae) form a large, pantropical clade of horticulturally important epiphytes. Monopodial leafless members of Vandeae have undergone extreme reduction in habit and represent a novel adaptation to the canopy environment in tropical Africa, Asia, and America. To study the evolution of monopodial leaflessness, molecular and structural evidence was used to generate phylogenetic hypotheses for Vandeae. Molecular analyses used sequence data from ITS nrDNA, trnL-F plastid DNA, and matK plastid DNA. Maximum parsimony analyses of these three DNA regions each supported two subtribes within monopodial Vandeae: Aeridinae and a combined Angraecinae + Aerangidinae. Adding structural characters to sequence data resulted in trees with more homoplasy, but gave fewer trees each with more well-supported clades than either data set alone. Two techniques for examining character evolution were compared: (1) mapping vegetative characters onto a molecular topology and (2) tracing vegetative characters onto a combined structural and molecular topology. In both cases, structural synapomorphies supporting monopodial Vandeae were nearly identical. A change in leaf morphology (usually reduced to a nonphotosynthetic scale), monopodial growth habit, and aeration complexes for gas exchange in photosynthetic roots seem to be the most important characters in making the evolutionary transition to leaflessness.

Floral convergence in Oncidiinae (Cymbidieae; Orchidaceae): an expanded concept of Gomesa and a new genus Nohawilliamsia

BACKGROUND Floral morphology, particularly the angle of lip attachment to the column, has historically been the fundamental character used in establishing generic limits in subtribe Oncidiinae (Orchidaceae), but it has also been long recognized that reliance on this character alone has produced a highly artificial set of genera. In essence, lip/column relationships reflect syndromes associated with pollinator preferences; most genera of Oncidiinae as previously defined have consisted of a single floral type. Here, the degree to which this has influenced generic delimitation in Brazilian members of the largest genus of Oncidiinae, Oncidium, which previous molecular (DNA) studies have demonstrated to be polyphyletic, is evaluated. METHODS Phylogenetic analyses of the following multiple DNA regions were used: the plastid psbA-trnH intergenic spacer, matK exon and two regions of ycf1 exon and nuclear ribosomal DNA, comprised of the two internal transcribed spacers, ITS1 and ITS2, and the 5.8S gene. Results from all regions analysed separately indicated highly similar relationships, so a combined matrix was analysed. KEY RESULTS Nearly all species groups of Brazilian Oncidium are only distantly related to the type species of the genus, O. altissimum, from the Caribbean. There are two exceptions to this geographical rule: O. baueri is related to the type group and O. orthostates, an isolated species that lacks the defining tabula infrastigmata of Oncidium, is not exclusively related to any previously described genus in the subtribe. Several well-supported subclades can be observed in these results, but they do not correspond well to sections of Oncidium as previously circumscribed or to segregate genera as defined by several recent authors. In spite of their floral differences, these groups of Oncidium, formerly treated as O. sections Barbata, Concoloria pro parte, Crispa, Ranifera, Rhinocerotes, Rostrata (only O. venustum), Synsepala, Verrucituberculata pro parte and Waluewa, form a well-supported clade with Gomesa (including Rodrigueziella and Rodrigueziopsis) embedded in it. Two often recognized segregate genera, Baptistonia and Ornithophora, and the recently described Carriella are also embedded within the Brazilian clade. The level of variation within major subclades of the Gomesa clade is low and similar to that observed within other genera of Oncidiinae. CONCLUSIONS Convergence on a stereotypical syndrome of floral traits associated with pollination by oil-collecting bees has resulted in these characters not being reliable for producing monophyletic taxa, and the genus Oncidium, defined by these characters, is grossly polyphyletic. Vegetative and a few floral/inflorescence characters link these taxa with a mainly Brazilian distribution, and they were all transferred to Gomesa on this basis rather than separated from Gomesa based on their floral differences, which we hypothesize to be simple shifts in pollination strategies. Other authors have described a large number of new genera for these former members of Oncidium, but most of these are not supported by the results presented here (i.e. they are not monophyletic). A new genus, Nohawilliamsia, is described for O. orthostates because it does not fit in any currently recognized genus and is only distantly related to any other member of Oncidiinae.

Function of glandular secretions in fragrance collection by male euglossine bees (Apidae: Euglossini).

MaleEulaema cingulata (Fabricius) (Apidae: Euglossini) possess large cephalic labial glands that secrete a mixture of lipids. In the process of fragrance collection, males secrete the labial gland lipids onto the substrate. The mixture of lipids and fragrances is then taken up by the front tarsal brushes and transferred to the hind tibial organs. The labial gland secretions apparently serve as a nonpolar solvent and carrier that increases the efficiency of fragrance collection.

Generic circumscription and relationships in the tribe Melanthieae (Liliales, Melanthiaceae), with emphasis on Zigadenus: evidence from ITS and trnL-F sequence data.

The circumscription and relationships of genera within the tribe Melanthieae (29 representative taxa) were evaluated using parsimony analyses of ITS (nuclear ribosomal) and trnL-F (plastid) DNA sequence data, alone and in combination. Proposed new generic circumscriptions, strongly supported by the tree statistics and topologies in all analyses, are correlated with potential morphological synapomorphies at the proper level of universality. Based on the molecular cladograms, Stenanthium is biphyletic, and the traditional Zigadenus s.l. (sensu lato) is polyphyletic. Amianthium and Schoenocaulon are distinct entities; the Veratrum complex is conservatively treated as one large monophyletic genus (including Melanthium). Although some generic relationships are not highly resolved, the analyses provide strong support for Zigadenus glaberrimus as sister to the rest of the tribe, and Amianthium muscitoxicum as closely related to Veratrum s.l. As a result of these analyses, seven genera (some with novel circumscription) are recognized within the tribe Melanthieae: Amianthium, Anticlea, Schoenocaulon, Stenanthium, Toxicoscordion, Veratrum, and Zigadenus.

Carvone Oxide: An Example of Convergent Evolution in Euglossine Pollinated Plants

Trans-carvone oxide has been found in the floral fragrances of nine species of Ca- tasetum, one species each of Aspasia and Notylia (all three genera in Orchidaceae), and in one species of Dalechampia (Euphorbiaceae). Carvone oxide is a major fragrance component in 11 of the 12 species analyzed, and has been synthesized and used in field bioassays in Panama, where it attracts five species of Eulaema and three species of Euglossa. We predict it will be a major attractant of Eulaema species that previously have not been attracted to chemical baits. We suggest that synthesis and secretion of carvone oxide by several genera of Orchidaceae and one species of Euphorbiaceae are the result of evolutionary convergence in floral rewards.