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.

Reproductive patterns, genetic diversity and inbreeding depression in two closely related Jumellea species with contrasting patterns of commonness and distribution.

BACKGROUNDS AND AIMS: Theory predicts that the long-term persistence of plant populations exposed to size reduction can be threatened by a loss of genetic diversity and increased inbreeding. However, several life-history and ecological traits can influence the response to population size reduction. The reproductive patterns, levels of genetic diversity and magnitude of inbreeding depression of the rare and fragmented Jumellea fragrans and of its widespread congener J. rossii were studied. The aim was to evaluate the effects of over-collection and fragmentation on J. fragrans and to enhance our knowledge of the biology and ecology of the two species, used for their aromatic and medicinal properties on Réunion. METHODS: Hand pollination experiments were conducted to determine the breeding system and to evaluate the potential for inbreeding depression in both species. Nuclear microsatellite markers were used to investigate selfing rates and levels of genetic diversity. KEY RESULTS: Jumellea rossii revealed a mixed-mating system, and inbreeding depression at the germination stage (δ = 0·66). Levels of genetic diversity were relatively high [allelic richness (AR) = 8·575 and expected heterozygosity (He) = 0·673]. In J. fragrans, selfing rates suggest a mainly outcrossing mating system. Genetic diversity was lower than in J. rossii, but not yet critically low (AR = 4·983 and He = 0·492), probably because of the mainly outcrossing mating system and the relatively high density of individuals in the studied population. Jumellea fragrans did not show inbreeding depression, and it is hypothesized that the population had progressively purged its genetic load during successive fragmentation events. CONCLUSIONS: Even if the persistence of the J. fragrans population is not threatened in the short term, its genetic diversity has probably been reduced by fragmentation and over-collection. In situ conservation actions for J. fragrans and ex situ cultivation of both species are recommended in order to meet the demand of local people.

Orchid conservation: making the links.

Orchidaceae, one of the largest families of flowering plants, present particular challenges for conservation, due in great part to their often complex interactions with mycorrhizal fungi, pollinators and host trees. In this Highlight, we present seven papers focusing on orchids and their interactions and other factors relating to their conservation.

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.

Phylogeny and generic limits in the sister tribes Psychotrieae and Palicoureeae (Rubiaceae): Evolution of schizocarps in Psychotria and origins of bacterial leaf nodules of the Malagasy species.

• Premise of the study: The pantropical, species-rich Psychotrieae and Palicoureeae are sister tribes of mostly drupe-bearing and nonbacterial leaf-nodulating species with problematic generic limits. This problem is more complicated in Psychotrieae due to the paraphyly of the genus Psychotria, the lack of diagnostic characters for some major lineages, and the poor sampling from some biodiversity hotspots. Schizocarps and bacterial leaf nodules have been used for recognizing formal groups in Psychotrieae, but their evolution and taxonomic value have not been studied using a robust phylogeny of the tribe.• Methods: We analyzed 287 samples from the entire ranges of the tribes, with particular emphasis on the Western Indian Ocean region, with the Bayesian Markov chain Monte Carlo method.• Key results: All allied Psychotria genera investigated are nested within a paraphyletic Psychotria. Schizocarps evolved independently two times within Psychotria, and one reversal back to the drupaceous condition is inferred. The Malagasy leaf-nodulated Psychotrieae (except Apomuria bullata) and the Comorian non-leaf-nodulated Psychotria conocarpa are nested within the (African) leaf-nodulated clade. Within Palicoureeae, Chassalia is paraphyletic with respect to Geophila sensu stricto, and the Malagasy Geophila gerrardii and the African Hymenocoleus are closely related.• Conclusions: A widely circumscribed Psychotria encompassing the entire Psychotrieae is supported. Within Psychotria, two separate origins of schizocarps from drupes, one reversal back to the drupaceous condition, and two independent origins of the Malagasy leaf-nodulated species are inferred. A new genus Puffia is described to accommodate Geophila gerrardii, and a narrow circumscription of Chassalia is adopted. Thirty-two new combinations, two lectotypifications, and 25 new names are presented.

Evidence for isolation-by-habitat among populations of an epiphytic orchid species on a small oceanic island.

Identifying factors that promote population differentiation is of interest for understanding the early stages of speciation. Gene flow among populations inhabiting different environments can be reduced by geographical distance (isolation-by-distance) or by divergent selection resulting from local adaptation (isolation-by-ecology). Few studies have investigated the influence of these factors in small oceanic islands where the influence of geographic distance is expected to be null but where habitat diversity could have a strong effect on population differentiation. In this study, we tested for the spatial divergence of phenotypes (floral morphology and floral scent) and genotypes (microsatellites) among ten populations of Jumellea rossii, an epiphytic orchid endemic to Réunion growing in three different habitats. We found a significant genetic differentiation between populations that is structured by habitat heterogeneity rather than by geographic distance between populations. These results suggest that ecological factors might reduce gene flow among populations located in different habitats. This pattern of isolation-by-habitat may be the result of both isolation-by-ecology by habitat filtering and asynchrony in flowering phenology. Furthermore, data on floral morphology match these findings, with multivariate analysis grouping populations by habitat type but could be only due to phenotypic plasticity. Indeed floral scent compounds were not significantly different between populations indicating that specific plant-pollinator mutualism does not seem to play a major role in the population differentiation of J. rossii. In conclusion, the results from our study emphasize the importance of habitat diversity of small oceanic islands as a factor of population differentiation.

The role of epiphytism in architecture and evolutionary constraint within mycorrhizal networks of tropical orchids.

Characterizing the architecture of bipartite networks is increasingly used as a framework to study biotic interactions within their ecological context and to assess the extent to which evolutionary constraint shape them. Orchid mycorrhizal symbioses are particularly interesting as they are viewed as more beneficial for plants than for fungi, a situation expected to result in an asymmetry of biological constraint. This study addressed the architecture and phylogenetic constraint in these associations in tropical context. We identified a bipartite network including 73 orchid species and 95 taxonomic units of mycorrhizal fungi across the natural habitats of Reunion Island. Unlike some recent evidence for nestedness in mycorrhizal symbioses, we found a highly modular architecture that largely reflected an ecological barrier between epiphytic and terrestrial subnetworks. By testing for phylogenetic signal, the overall signal was stronger for both partners in the epiphytic subnetwork. Moreover, in the subnetwork of epiphytic angraecoid orchids, the signal in orchid phylogeny was stronger than the signal in fungal phylogeny. Epiphytic associations are therefore more conservative and may co-evolve more than terrestrial ones. We suggest that such tighter phylogenetic specialization may have been driven by stressful life conditions in the epiphytic niches. In addition to paralleling recent insights into mycorrhizal networks, this study furthermore provides support for epiphytism as a major factor affecting ecological assemblage and evolutionary constraint in tropical mycorrhizal symbioses.

Colour-scent associations in a tropical orchid: three colours but two odours.

Colour and scent are the major pollinator attractants to flowers, and their production may be linked by shared biosynthetic pathways. Species with polymorphic floral traits are particularly relevant to study the joint evolution of floral traits. We used in this study the tropical orchid Calanthe sylvatica from Réunion Island. Three distinct colour varieties are observed, presenting lilac, white or purple flowers, and named respectively C. sylvaticavar.lilacina (hereafter referred as var. lilacina), C. sylvaticavar. alba (var. alba) and C. sylvatica var. purpurea (var. purpurea). We investigated the composition of the floral scent produced by these colour varieties using the non-invasive SPME technique in the wild. Scent emissions are dominated by aromatic compounds. Nevertheless, the presence of the terpenoid (E)-4,8-dimethylnona-1,3,7-triène (DMNT) is diagnostic of var. purpurea, with the volatile organic compounds (VOC) produced by some individuals containing up to 60% of DMNT. We evidence specific colour-scent associations in C. sylvatica, with two distinct scent profiles in the three colour varieties: the lilacina-like profile containing no or very little DMNT (<2%) and the purpurea-like profile containing DMNT (>2%). Calanthe sylvatica var. alba individuals group with one or the other scent profile independently of their population of origin. We suggest that white-flowered individuals have evolved at least twice, once from var. lilacina and at least once from var. purpurea after the colonisation of la Réunion. White-flowered individuals may have been favoured by the particular pollinator fauna characterising the island. These flowering varieties of C. sylvatica, which display three colours but two scents profiles prove that colour is not always a good indicator of odour and that colour-scent associations may be complex, depending on pollination ecology of the populations concerned.

Saprotrophic fungal mycorrhizal symbionts in achlorophyllous orchids: finding treasures among the 'molecular scraps'?

Mycoheterotrophic plants are achlorophyllous plants that obtain carbon from their mycorrhizal fungi. They are usually considered to associate with fungi that are (1) specific of each mycoheterotrophic species and (2) mycorrhizal on surrounding green plants, which are the ultimate carbon source of the entire system. Here we review recent works revealing that some mycoheterotrophic plants are not fungal-specific, and that some mycoheterotrophic orchids associate with saprophytic fungi. A re-examination of earlier data suggests that lower specificity may be less rare than supposed in mycoheterotrophic plants. Association between mycoheterotrophic orchids and saprophytic fungi arose several times in the evolution of the two partners. We speculate that this indirectly illustrates why transition from saprotrophy to mycorrhizal status is common in fungal evolution. Moreover, some unexpected fungi occasionally encountered in plant roots should not be discounted as 'molecular scraps', since these facultatively biotrophic encounters may evolve into mycorrhizal symbionts in some other plants.

Orthoptera, a new order of pollinator.

BACKGROUND AND AIMS: Pollinator-mediated selection and evolution of floral traits have long fascinated evolutionary ecologists. No other plant family shows as wide a range of pollinator-linked floral forms as Orchidaceae. In spite of the large size of this model family and a long history of orchid pollination biology, the identity and specificity of most orchid pollinators remains inadequately studied, especially in the tropics where the family has undergone extensive diversification. Angraecum (Vandeae, Epidendroideae), a large genus of tropical Old World orchids renowned for their floral morphology specialized for hawkmoth pollination, has been a model system since the time of Darwin. METHODS: The pollination biology of A. cadetii, an endemic species of the islands of Mauritius and Reunion (Mascarene Islands, Indian Ocean) displaying atypical flowers for the genus (white and medium-size, but short-spurred) was investigated. Natural pollinators were observed by means of hard-disk camcorders. Pollinator-linked floral traits, namely spur length, nectar volume and concentration and scent production were also investigated. Pollinator efficiency (pollen removal and deposition) and reproductive success (fruit set) were quantified in natural field conditions weekly during the 2003, 2004 and 2005 flowering seasons (January to March). KEY RESULTS: Angraecum cadetii is self-compatible but requires a pollinator to achieve fruit set. Only one pollinator species was observed, an undescribed species of raspy cricket (Gryllacrididae, Orthoptera). These crickets, which are nocturnal foragers, reached flowers by climbing up leaves of the orchid or jumping across from neighbouring plants and probed the most 'fresh-looking' flowers on each plant. Visits to flowers were relatively long (if compared with the behaviour of birds or hawkmoths), averaging 16.5 s with a maximum of 41.0 s. At the study site of La Plaine des Palmistes (Pandanus forest), 46.5 % of flowers had pollen removed and 27.5 % had pollinia deposited on stigmas. The proportion of flowers that set fruit ranged from 11.9 % to 43.4 %, depending of the sites sampled across the island. CONCLUSIONS: Although orthopterans are well known for herbivory, this represents the first clearly supported case of orthopteran-mediated pollination in flowering plants.

Independent recruitment of saprotrophic fungi as mycorrhizal partners by tropical achlorophyllous orchids.

Mycoheterotrophic orchids have adapted to shaded forest understory by shifting to achlorophylly and receiving carbon from their mycorrhizal fungi. In temperate forests, they associate in a highly specific way with fungi forming ectomycorrhizas on nearby trees, and exploiting tree photosynthates. However, many rainforests lack ectomycorrhizal fungi, and there is evidence that some tropical Asiatic species associate with saprotrophic fungi. To investigate this in different geographic and phylogenetic contexts, we identified the mycorrhizal fungi supporting two tropical mycoheterotrophic orchids from Mascarene (Indian Ocean) and Caribbean islands. We tested their possible carbon sources by measuring natural nitrogen ((15)N) and carbon ((13)C) abundances. Saprotrophic basidiomycetes were found: Gastrodia similis associates with a wood-decaying Resinicium (Hymenochaetales); Wullschlaegelia aphylla associates with both litter-decaying Gymnopus and Mycena species, whose rhizomorphs link orchid roots to leaf litter. The (15)N and (13)C abundances make plausible food chains from dead wood to G. similis and from dead leaves to W. aphylla. We propose that temperature and moisture in rainforests, but not in most temperate forests, may favour sufficient saprotrophic activity to support development of mycoheterotrophs. By enlarging the spectrum of mycorrhizal fungi and the level of specificity in mycoheterotrophic orchids, this study provides new insights on orchid and mycorrhizal biology in the tropics.

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.

Bird pollination in an angraecoid orchid on Reunion Island (Mascarene Archipelago, Indian Ocean).

BACKGROUND AND AIMS: Although numerous angraecoid orchids in Madagascar display typical sphingophilous syndrome (i.e. white, nectariferous, long-spurred flowers, producing a strong scent at the crepuscule that is attractive to moths), three species of Angraecum in Reunion, belonging to the endemic section Hadrangis, have atypical unscented and short-spurred flowers. The aim of the study was to investigate the implication of plant-pollinator interaction on the evolution of floral morphology of these peculiar island floral forms. METHODS: The flower morphology of A. striatum (one of the three section Hadrangis species) was investigated by performing a set of floral measures, and the reproductive biology was investigated by a set of hand pollination experiments. Natural pollinators were observed by means of a digital video camera. Pollinator efficiency (pollen removal and deposition) and reproductive success (fruit set) were quantified once a week in natural field conditions during the 2005 flowering season (i.e. from January to March). KEY RESULTS: The orchid is self-compatible but requires a pollinator to achieve fruit set. Only one pollinator was observed, the endemic white-eye Zosterops borbonicus (Zosteropidae). These birds perched on inflorescences, and probed most fresh-looking flowers on each plant for nectar. Nectar was both abundant (averaging 7.7 microL) and dilute (averaging 9.7 % sugar in sucrose equivalents). Birds were mostly active between 0830 and 0930 h. Visits to plants were extremely short, lasting from 9 to 27 s. At the study site, 60.9 % of flowers had pollen removed, and 46.4 % had pollinia deposited on stigmas. The proportion of flowers that initiated a fruit averaged 20.6 % in natural conditions. CONCLUSIONS: For the first time, a bird-pollinated orchid is described from a sub-tribe that is mainly specialized for moth pollination. This study documents a morphological shift in flowers in response to pollinator adaptations in the insular context of the Mascarene Archipelago.