Sexual deception of a beetle pollinator through floral mimicry.

Sexual mimicry is a complex multimodal strategy used by some plants to lure insects to flowers for pollination.1-4 It is notable for being highly species-specific and is typically mediated by volatiles belonging to a restricted set of chemical compound classes.3,4 Well-documented cases involve exploitation of bees and wasps (Hymenoptera)5,6 and flies (Diptera).7-9 Although beetles (Coleoptera) are the largest insect order and are well known as pollinators of both early and modern plants,10,11 it has been unclear whether they are sexually deceived by plants during flower visits.12,13 Here we report the discovery of an unambiguous case of sexual deception of a beetle: male longhorn beetles (Chorothyse hessei, Cerambycidae) pollinate the elaborate insectiform flowers of a rare southern African orchid (Disa forficaria), while exhibiting copulatory behavior including biting the antennae-like petals, curving the abdomen into the hairy lip cleft, and ejaculating sperm. The beetles are strongly attracted by (16S,9Z)-16-ethyl hexadec-9-enolide, a novel macrolide that we isolated from the floral scent. Structure-activity studies14,15 confirmed that chirality and other aspects of the structural geometry of the macrolide are critical for the attraction of the male beetles. These results demonstrate a new biological function for plant macrolides and confirm that beetles can be exploited through sexual deception to serve as pollinators.

Phylogenetic relationships amongst the African genera of subtribe Orchidinae s.l. (Orchidaceae; Orchideae): Implications for subtribal and generic delimitations.

Phylogenetic relationships within the Orchideae sensu Pridgeon et al, remain one of the biggest unresolved issues in our understanding of the taxonomy of the orchids. Members of the Orchideae are numerous and widespread in Africa but remain poorly represented in phylogenetic research. In this study we included a broad sampling of African taxa for which we sequenced three plastid (rbcl, matK and trnL + trnL-F) and two nuclear regions (ITS and 18S). We used 368 sequences representing 278 species and 49 genera to infer relationships using the Bayesian Inference and Maximum Likelihood method. Our results show strong support for three clades, two of which almost entirely match the historical circumscription of Orchidinae and Habenariinae, and the third, Bartholininae, sister to the former two, includes the genera Holothrix and Bartholina. Stenoglottis should be assigned to Orchidinae and not to Habenariinae. Several genera such as Habenaria, Cynorkis and Benthamia are shown to be para- or polyphyletic: Bonatea, Centrostigma, Platycoryne and Roeperocharis are all embedded in Habenaria; Physoceras, Arnottia and part of Benthamia are embedded in Cynorkis. We propose a subdivision of Orchideae sensu lato into nine subtribes, but refrain from making generic re-arrangements until more extensive or more in-depth studies have been done.

Trade in Zambian Edible Orchids-DNA Barcoding Reveals the Use of Unexpected Orchid Taxa for Chikanda.

In Zambia, wild edible terrestrial orchids are used to produce a local delicacy called chikanda, which has become increasingly popular throughout the country. Commercialization puts orchid populations in Zambia and neighbouring countries at risk of overharvesting. Hitherto, no study has documented which orchid species are traded on local markets, as orchid tubers are difficult to identify morphologically. In this study, the core land-plant DNA barcoding markers rbcL and matK were used in combination with nrITS to determine which species were sold in Zambian markets. Eighty-two interviews were conducted to determine harvesting areas, as well as possible sustainability concerns. By using nrITS DNA barcoding, a total of 16 orchid species in six different genera could be identified. Both rbcL and matK proved suitable to identify the tubers up to the genus or family level. Disa robusta, Platycoryne crocea and Satyrium buchananii were identified most frequently and three previously undocumented species were encountered on the market. Few orchid species are currently listed on the global International Union for the Conservation of Nature (IUCN) Red List. Local orchid populations and endemic species could be at risk of overharvesting due to the intensive and indiscriminate harvesting of chikanda orchids, and we therefore encourage increased conservation assessment of terrestrial African orchids.

New phylogenetic insights toward developing a natural generic classification of African angraecoid orchids (Vandeae, Orchidaceae).

Despite significant progress made in recent years toward developing an infrafamilial classification of Orchidaceae, our understanding of relationships among and within tribal and subtribal groups of epidendroid orchids remains incomplete. To reassess generic delimitation among one group of these epidendroids, the African angraecoids, phylogenetic relationships were inferred from DNA sequence data from three regions, ITS, matK, and the trnL-trnF intergenic spacer, obtained from a broadly representative sample of taxa. Parsimony and Bayesian analyses yielded highly resolved trees that are in clear agreement and show significant support for many key clades within subtribe Angraecinae s.l. Angraecoid orchids comprise two well-supported clades: an African/American group and an Indian Ocean group. Molecular results also support many previously proposed relationships among genera, but also reveal some unexpected relationships. The genera Aerangis, Ancistrorhynchus, Bolusiella, Campylocentrum, Cyrtorchis, Dendrophylax, Eurychone, Microcoelia, Nephrangis, Podangis and Solenangis are all shown to be monophyletic, but Angraecopsis, Diaphananthe and Margelliantha are polyphyletic. Diaphananthe forms three well-supported clades, one of which might represent a new genus, and Rhipidoglossum is paraphyletic with respect to Cribbia and Rhaesteria, and also includes taxa currently assigned to Margelliantha. Tridactyle too is paraphyletic as Eggelingia is embedded within it. The large genus Angraecum is confirmed to be polyphyletic and several groups will have to be recognized as separate genera, including sections Dolabrifolia and Hadrangis. The recently segregated genus Pectinariella (previously recognized as A. sect. Pectinaria) is polyphyletic and its Continental African species will have to be removed. Similarly, some of the species recently transferred to Angraecoides that were previously placed in Angraecum sects. Afrangraecum and Conchoglossum will have to be moved and described as a new genus.

A well-sampled phylogenetic analysis of the polystichoid ferns (Dryopteridaceae) suggests a complex biogeographical history involving both boreotropical migrations and recent transoceanic dispersals.

Intercontinental disjunctions in ferns have often been considered as the result of long-distance dispersal (LDD) events rather than of vicariance. However, in many leptosporangiate groups, both processes appear to have played a major role in shaping current geographical distribution. In this study, we reconstructed the phylogenetic relationships and inferred the ancestral distribution areas of the polystichoid ferns (Cyrtomium, Phanerophlebia, and Polystichum), to evaluate the relative impact of vicariance and LDD on the biogeography of this group. We used a molecular dataset including 3346 characters from five plastid loci. With 190 accessions our taxon coverage was about three times as large as any previous worldwide sampling. Biogeographical analyses were performed using S-DIVA and S-DEC and divergence times were estimated by integrating fossil and secondary calibrations. The polystichoid ferns are a monophyletic clade that may have originated in East Asia during the Eocene, an age much younger than previously estimated. Three transoceanic disjunctions between East Asia and New World were identified in the Paleogene: one for Phanerophlebia during late Eocene (34Ma, 19-51Ma), and two in Polystichum at the Eocene-Oligocene boundary (30Ma, 18-43Ma; 28Ma, 19-39Ma respectively). During the Neogene, further range expansions took place from Asia to Africa, Hawaii, and the Southwestern Indian Ocean region. Our results indicate that early transfers between the Old and the New World are compatible with a boreotropical migration scenario. After evolving in Asia during the Eocene, the polystichoid ferns reached the New World in independent migrations at the Eocene-Oligocene boundary through the boreotropical belt. However, although less likely, the alternative hypothesis of independent transoceanic dispersals from the Old to the New World cannot be ruled out. Further range expansion during the Neogene was most likely the result of long-distance dispersal (LDD).

Chemical and morphological filters in a specialized floral mimicry system.

Many plant species attract insect pollinators through chemical mimicry of their oviposition sites, often detaining them in a trap chamber that ensures pollen transfer. These plant mimics are considered to be unspecialized at the pollinator species level, yet field observations of a mycoheterotrophic rainforest orchid (Gastrodia similis), which emits an odour reminiscent of rotting fruit, indicate that it is pollinated by a single drosophilid fly species (Scaptodrosophila bangi). We investigated the roles of floral volatiles and the dimensions of the trap chamber in enforcing this specialization, using gas chromatography-mass spectrometry analyses, bioassays and scanning electron microscopy. We showed that G. similis flowers predominantly emit three fatty-acid esters (ethyl acetate, ethyl isobutyrate and methyl isobutyrate) that were shown in experiments to attract only Scaptodrosophila flies. We additionally showed that the trap chamber, which flies enter into via a touch-sensitive 'trapdoor', closely matches the body size of the pollinator species S. bangi and plays a key role in pollen transfer. Our study demonstrates that specialization in oviposition site mimicry is due primarily to volatile chemistry and is reflected in the dimensions of the trapping apparatus. It also indicates that mycoheterotrophic plants can be specialized both on mycorrhizal fungi and insect pollinators.

The evolution of floral nectaries in Disa (Orchidaceae: Disinae): recapitulation or diversifying innovation?

BACKGROUND AND AIMS: The Orchidaceae have a history of recurring convergent evolution in floral function as nectar production has evolved repeatedly from an ancestral nectarless state. However, orchids exhibit considerable diversity in nectary type, position and morphology, indicating that this convergence arose from alternative adaptive solutions. Using the genus Disa, this study asks whether repeated evolution of floral nectaries involved recapitulation of the same nectary type or diversifying innovation. Epidermis morphology of closely related nectar-producing and nectarless species is also compared in order to identify histological changes that accompanied the gain or loss of nectar production. METHODS: The micromorphology of nectaries and positionally equivalent tissues in nectarless species was examined with light and scanning electron microscopy. This information was subjected to phylogenetic analyses to reconstruct nectary evolution and compare characteristics of nectar-producing and nectarless species. KEY RESULTS: Two nectary types evolved in Disa. Nectar exudation by modified stomata in floral spurs evolved twice, whereas exudation by a secretory epidermis evolved six times in different perianth segments. The spur epidermis of nectarless species exhibited considerable micromorphological variation, including strongly textured surfaces and non-secreting stomata in some species. Epidermis morphology of nectar-producing species did not differ consistently from that of rewardless species at the magnifications used in this study, suggesting that transitions from rewardlessness to nectar production are not necessarily accompanied by visible morphological changes but only require sub-cellular modification. CONCLUSIONS: Independent nectary evolution in Disa involved both repeated recapitulation of secretory epidermis, which is present in the sister genus Brownleea, and innovation of stomatal nectaries. These contrasting nectary types and positional diversity within types imply weak genetic, developmental or physiological constraints in ancestral, nectarless Disa. Such functional convergence generated by morphologically diverse solutions probably also underlies the extensive diversity of nectary types and positions in the Orchidaceae.

Ancestral deceit and labile evolution of nectar production in the African orchid genus Disa.

An outstanding feature of the orchid family is that approximately 30-40% of the species have non-rewarding flowers and deploy various modes of deception to attract pollinators, whereas the remaining species engage in pollination mutualisms based on provision of floral rewards. Here, we explore the direction, frequency and reversibility of transitions between deceptive and rewarding pollination systems in the radiation of the large African genus Disa, and test whether these transitions had consequences for diversification. By optimizing nectar production data for 111 species on a well-resolved phylogeny, we confirmed that floral deception was the ancestral condition and that nectar production evolved at least nine times and was lost at least once. Transitions to nectar production first occurred ca 17 million years ago but did not significantly affect either speciation or extinction rates. Nectar evolved independently of a spur, which was lost and gained multiple times. These results show that nectar production can be a highly labile trait and highlight the need for further studies of the genetic architecture of nectar production and the selective factors underlying transitions between deception and mutualism.

Extinction risk and diversification are linked in a plant biodiversity hotspot.

It is widely recognized that we are entering an extinction event on a scale approaching the mass extinctions seen in the fossil record. Present-day rates of extinction are estimated to be several orders of magnitude greater than background rates and are projected to increase further if current trends continue. In vertebrates, species traits, such as body size, fecundity, and geographic range, are important predictors of vulnerability. Although plants are the basis for life on Earth, our knowledge of plant extinctions and vulnerabilities is lagging. Here, we disentangle the underlying drivers of extinction risk in plants, focusing on the Cape of South Africa, a global biodiversity hotspot. By comparing Red List data for the British and South African floras, we demonstrate that the taxonomic distribution of extinction risk differs significantly between regions, inconsistent with a simple, trait-based model of extinction. Using a comprehensive phylogenetic tree for the Cape, we reveal a phylogenetic signal in the distribution of plant extinction risks but show that the most threatened species cluster within short branches at the tips of the phylogeny--opposite to trends in mammals. From analyzing the distribution of threatened species across 11 exemplar clades, we suggest that mode of speciation best explains the unusual phylogenetic structure of extinction risks in plants of the Cape. Our results demonstrate that explanations for elevated extinction risk in plants of the Cape flora differ dramatically from those recognized for vertebrates. In the Cape, extinction risk is higher for young and fast-evolving plant lineages and cannot be explained by correlations with simple biological traits. Critically, we find that the most vulnerable plant species are nonetheless marching towards extinction at a more rapid pace but, surprisingly, independently from anthropogenic effects. Our results have important implications for conservation priorities and cast doubts on the utility of current Red List criteria for plants in regions such as the Cape, where speciation has been rapid, if our aim is to maximize the preservation of the tree-of-life.

Consistent phenological shifts in the making of a biodiversity hotspot: the Cape flora.

BACKGROUND: The best documented survival responses of organisms to past climate change on short (glacial-interglacial) timescales are distributional shifts. Despite ample evidence on such timescales for local adaptations of populations at specific sites, the long-term impacts of such changes on evolutionary significant units in response to past climatic change have been little documented. Here we use phylogenies to reconstruct changes in distribution and flowering ecology of the Cape flora--South Africa's biodiversity hotspot--through a period of past (Neogene and Quaternary) changes in the seasonality of rainfall over a timescale of several million years. RESULTS: Forty-three distributional and phenological shifts consistent with past climatic change occur across the flora, and a comparable number of clades underwent adaptive changes in their flowering phenology (9 clades; half of the clades investigated) as underwent distributional shifts (12 clades; two thirds of the clades investigated). Of extant Cape angiosperm species, 14-41% have been contributed by lineages that show distributional shifts consistent with past climate change, yet a similar proportion (14-55%) arose from lineages that shifted flowering phenology. CONCLUSIONS: Adaptive changes in ecology at the scale we uncover in the Cape and consistent with past climatic change have not been documented for other floras. Shifts in climate tolerance appear to have been more important in this flora than is currently appreciated, and lineages that underwent such shifts went on to contribute a high proportion of the flora's extant species diversity. That shifts in phenology, on an evolutionary timescale and on such a scale, have not yet been detected for other floras is likely a result of the method used; shifts in flowering phenology cannot be detected in the fossil record.

Estimating the age of fire in the Cape flora of South Africa from an orchid phylogeny.

Fire may have been a crucial component in the evolution of the Cape flora of South Africa, a region characterized by outstanding levels of species richness and endemism. However, there is, to date, no critical assessment of the age of the modern fire regime in this biome. Here, we exploit the presence of two obligate post-fire flowering clades in the orchid genus Disa, in conjunction with a robust, well-sampled and dated molecular phylogeny, to estimate the age by which fire must have been present. Our results indicate that summer drought (winter rainfall), the fire regime and the fynbos vegetation are several million years older than currently suggested. Summer drought and the fynbos vegetation are estimated to date back to at least the Early Miocene (ca 19.5 Ma). The current fire regime may have been established during a period of global cooling that followed the mid-Miocene Climatic Optimum (ca 15 Ma), which led to the expansion of open habitats and increased aridification. The first appearance of Disa species in the grassland biome, as well as in the subalpine habitat, is in striking agreement with reliable geological and palaeontological evidence of the age of these ecosystems, thus corroborating the efficacy of our methods. These results change our understanding of the historical mechanisms underlying botanical evolution in southern Africa, and confirm the potential of using molecular phylogenies to date events for which other information is lacking or inconclusive.

Phylogenetics and biogeography of Mascarene angraecoid orchids (Vandeae, Orchidaceae).

The large angraecoid orchid clade (subtribe Angraecinae sensu lato) has undergone extensive radiation in the western Indian Ocean, which includes Africa, Madagascar, and a number of Indian Ocean islands, such as the Mascarene Archipelago. To investigate systematics and biogeography of these Mascarene orchids, phylogenetic relationships were inferred from four plastid DNA regions, trnL intron, trnL-F intergenic spacer, matK gene, and rps16 intron. Parsimony and Bayesian analyses provided identical sets of relationships within the subtribe; the large genus Angraecum as currently circumscribed does not form an exclusive clade. Bonniera, an endemic genus to Reunion, is shown to be embedded in part of Angraecum. Evidence from our research supports the main origin of Mascarene Angraecinae from Madagascar, and although there were many independent colonizations, only a few of the lineages radiated within the Mascarene Archipelago.

The Cape element in the Afrotemperate flora: from Cape to Cairo?

The build-up of biodiversity is the result of immigration and in situ speciation. We investigate these two processes for four lineages (Disa, Irideae p.p., the Pentaschistis clade and Restionaceae) that are widespread in the Afrotemperate flora. These four lineages may be representative of the numerous clades which are species rich in the Cape and also occur in the highlands of tropical Africa. It is as yet unclear in which direction the lineages spread. Three hypotheses have been proposed: (i) a tropical origin with a southward migration towards the Cape, (ii) a Cape origin with a northward migration into tropical Africa, and (iii) vicariance. None of these hypotheses has been thoroughly tested. We reconstruct the historical biogeography of the four lineages using likelihood optimization onto molecular phylogenies. We find that tropical taxa are nested within a predominantly Cape clade. There is unidirectional migration from the Cape into the Drakensberg and from there northwards into tropical Africa. The amount of in situ diversification differs between areas and clades. Dating estimates show that the migration into tropical East Africa has occurred in the last 17 Myr, consistent with the Mio-Pliocene formation of the mountains in this area.

A molecular phylogeny for the large African orchid genus Disa.

Phylogenetic relationships were inferred for the African subtribe Disinae (Orchidoideae, Orchidaceae), which include the large genus Disa and the small genus Schizodium. One nuclear (ITS) gene region and two plastid (trnLF and matK) gene regions were sequenced for 136 ingroup, representing 70% of all known Disinae species, as well as for 7 outgroup taxa. The combined data matrix contained 4094 characters and was analysed using parsimony and Bayesian inference. Our results show that the generic status of Schizodium can no longer be supported, as it is deeply embedded within the genus Disa. Furthermore, the currently recognised subgenera do not reflect the phylogenetic relationships and should be rejected. Several of the currently recognised sections are monophyletic, others contain misplaced elements, while some are polyphyletic. Morphological divergence, rather than convergence, has hampered previous attempts at a phylogenetic classification of the Disinae. On the basis of our molecular phylogenetic hypothesis, we propose a monotypic subtribe Disinae and a subdivision of the genus Disa into 18 sections.

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.