Claire Micheneau

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.

Auto-pollination in a long-spurred endemic orchid (Jumellea stenophylla) on Reunion Island (Mascarene Archipelago, Indian Ocean)

SummarySince Darwin, long-spurred angraecoid orchids have been known for their fascinating evolutionary relationship with long-tongued hawkmoths (Sphingidae) on Madagascar. We studied the reproductive biology of the long-spurred endemic Jumellea stenophylla on Reunion. Despite the species exhibits flowers with the typical sphingophilous pollination syndrome (i.e. spur length averaged 137.9 mm, mean nectar volume was 6.1 µl, and nectar concentration was 10.7% sugar in sucrose equivalent), it does not require pollinators to achieve fruits. Compared with other hawkmoth-pollinated orchids, flower longevity was very short, lasting less than 5 days, and the species did not emit the characteristic strong and sweet scent at dusk. Fruit set ranged from 66.7 to 83.9% when pollinators were excluded, and 56–77.5% under natural conditions. Auto-pollination is a consequence of structural modifications. On Reunion, such breeding system is not rare within long-spurred species, and seems linked to the absence of specific pollinator during island colonization, and species establishment.

Development and characterization of microsatellite loci in Pericopsis elata (Fabaceae) using a cost-efficient approach.

PREMISE OF THE STUDY Microsatellite loci were developed in the endangered Pericopsis elata using a combination of low-cost procedures. METHODS AND RESULTS Microsatellite isolation was performed simultaneously on three distinct species through a newly available procedure that associates multiplex microsatellite enrichment and next-generation sequencing, allowing the rapid and low-cost development of microsatellite-enriched libraries through the use of a 1/32nd GS-FLX plate. Genotyping using M13-like labeling in multiplexed reactions allowed additional cost savings. From 72 primers selected for initial screening, 21 positively amplified P. elata, and 11 showed polymorphism with two to 11 alleles per locus and a mean value of 5.4 alleles per locus. CONCLUSIONS These microsatellite loci will be useful to further investigate the level of genetic variation within and between natural populations of P. elata in Africa.

Sapromyiophily in the native orchid, Bulbophyllum variegatum , on Réunion (Mascarene Archipelago, Indian Ocean)

Orchid species are well known for their highly specialized pollinator interactions. To better understand the reproductive biology of the tropical epiphytic orchid Bulbophyllum variegatum on Reunion, we investigated the floral morphology, breeding system, pollinator diversity, floral scent profile and fruiting success of about 30 individuals in three natural populations during two consecutive flowering seasons. Controlled hand-pollination experiments in two populations showed that the species is self-compatible, but requires pollinator service to achieve reproduction. Videotape pollinator observations were conducted during two flowering seasons for 56 h and revealed that B. variegatum is pollinated by a single species of fly from the Platystomatidae. This fly seems to be attracted by the unpleasant scent produced by the flowers, and does not receive any reward after achieving pollination. In addition, no egg-laying behaviour was observed. Bulbophyllum variegatum thus exhibits a typical sapromyiophilous pollination syndrome which constitutes the first proven case of sapromyiophily within the genus Bulbophyllum on the Mascarene Archipelago. Hand pollinations further showed that fruit set was not significantly higher for flowers that received outcross pollen than for those that were self-crossed (53% and 44% respectively). Fruit sets under natural conditions were significantly different among populations, ranging from 0.5% to 24.3%. This low fruit production is likely due to infrequent pollinator visits, particularly in disturbed forests where the pollinator has never been observed.

Orchid-bird interactions: a case study from Angraecum (Vandeae, Angraecinae) and Zosterops (white-eyes, Zosteropidae) on Reunion Island

Although angraecoid orchids are famous for their spectacular flowers, pollinated by hawkmoths in Madagascar, two species of Angraecum endemic to the Mascarenes (sect. Hadrangis ) have atypical unscented and short-spurred flowers that are pollinated by birds. We focused on the pollination and reproductive success of Angraecum bracteosum Balf.f. & S.Moore, a member of sect. Hadrangis . We investigated the breeding system by performing a set of hand-pollination ex- periments and by observing natural pollinators using video-captures. Pollinator efficiency (pollen removal and deposition) and reproductive success (fruit set) were quantified once a week in two natural island populations during three consecutive flowering seasons (2003–2005). The orchid is self-compatible but requires pollinator services to achieve reproduction. The two white-eye song-bird species (Zosteropidae) that occur on Reunion, Zosterops borbonicus Boddaert and Zosterops oliva- Ceus L., were recorded as pollinators. However, while pollination events were rare and occasional following flower visits by Z. borbonicus , foraging of Z. oliveaceus resulted in a high frequency of successful pollinations. White-eyes typically perched on inflorescences, and probed most of the ‘‘fresh-looking’’ flowers for nectar. This study documents a probable case of bird-pollination specialization within Angraecum section Hadrangis , and sheds light on the role played by pollina- tors during the radiation of section Hadrangis in the Mascarene Archipelago.

Morphometrics and molecular phylogenetics of Angraecum section Dolabrifolia (Orchidaceae, Angraecinae)

Species delimitation within Angraecum section Dolabrifolia is problematic due to morphological variability coupled with overlap in many of the characters traditionally used to distinguish species. Recent molecular phylogenetic studies of the genus included three of the five currently described species of the Dolabrifolia group, placing them as sister to continental African species of Angraecum sect. Pectinaria. In preparation for a taxonomic revision of section Dolabrifolia, we analyzed morphological and molecular data to re-assess the circumscription of each of the five currently described species, examined the relationships among members of the section, and assessed their position within the genus. We used 172 alcohol-preserved specimens to perform multivariate analyses on 15 morphological characters. We also collected molecular sequence data from 16 taxa including all members of the section using six DNA regions, and analyzed these data with parsimony and Bayesian methods. The morphometric study revealed five distinct groups, of which four correspond to currently recognized species, while the fifth represents a taxonomic novelty. Angraecum podochiloides is the most distinctive morphologically, recognizable by its narrow leaves bearing white-yellowish flowers. The often confused species Angraecum distichum and Angraecum bancoense are clearly distinguishable by flower size. Molecular phylogenetic analyses indicated that section Dolabrifolia forms a well-supported clade related to the continental African members of section Pectinaria. Four species are well delimited, while the accessions of Angraecum aporoides formed two well-supported clades corresponding to two subclusters revealed by the morphometric study. The recently published Angraecum poppendickianum is shown to be a synonym of A. distichum.

Taxonomic revision of the continental African species of Angraecum section Pectinaria (Orchidaceae)

Abstract Angraecum, the largest genus of the angraecoid orchids, has long been regarded as a natural group possessing a common overall floral morphology, although recent molecular studies have called into question its monophyly. No recent taxonomic revision is available for the genus or its sections, and previous studies (whether based on morphology or DNA sequences) have suffered from a paucity of material from continental Africa. An earlier study suggested that the section Pectinaria was polyphyletic, with one clade, containing the type species, centered in Madagascar and the other in continental Africa. Our recent morphometric and molecular study confirmed the polyphyly of this section, clarified the circumscription of its continental African species, and assessed their monophyly. Here we present a comprehensive taxonomic revision of the five continental African members of Angraecum section Pectinaria, including an identification key, distribution maps, and an assessment of their conservation status (two are threatened, one could become threatened in the near future, and two do not meet the criteria for threatened status). Species richness is highest in Atlantic Central Africa, and A. gabonense is reported for the first time from Equatorial Guinea. Lectotypifications are made for A. pungens, A. subulatum and its synonym, A. canaliculatum.

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.

Australasian orchid biogeography at continental scale: Molecular phylogenetic insights from the Sun Orchids (Thelymitra, Orchidaceae)

Australia harbours a rich and highly endemic orchid flora, with c. 90% of species endemic to the country. Despite that, the biogeographic history of Australasian orchid lineages is only poorly understood. Here we examined evolutionary relationships and the spatio-temporal evolution of the sun orchids (Thelymitra, 119 species), which display disjunct distribution patterns frequently found in Australasian orchid lineages. Phylogenetic analyses were conducted based on one nuclear (ITS) and three plastid markers (matK, psbJ-petA, ycf1) using Maximum Likelihood and Bayesian inference. Divergence time estimations were carried out with a relaxed molecular clock in a Bayesian framework. Ancestral ranges were estimated using the dispersal-extinction-cladogenesis model and an area coding based on major disjunctions. The phylogenetic analyses clarified intergeneric relationships within Thelymitrinae, with Epiblema being sister to Thelymitra plus Calochilus, both of which were well-supported. Within Thelymitra, eight major and several minor clades were retrieved in the nuclear and plastid phylogenetic reconstructions. Five major clades corresponded to species complexes previously recognized based on morphological characters, whereas other previously recognized species groups were found to be paraphyletic. Conflicting signals between the nuclear and plastid phylogenetic reconstructions provided support for hybridization and plastid capture events both in the deeper evolutionary history of the genus and more recently. Divergence time estimation placed the origin of Thelymitra in the late Miocene (c. 10.8 Ma) and the origin of the majority of the main clades within Thelymitra during the late Pliocene and early Pleistocene, with the majority of extant species arising during the Pleistocene. Ancestral range reconstruction revealed that the early diversification of the genus in the late Miocene and Pliocene took place predominantly in southwest Australia, where most species with highly restricted distributional ranges occur. Several long-distance dispersal events eastwards across the Nullarbor Plain were inferred, recurrently resulting in lineage divergence within the genus. The predominant eastwards direction of long-distance dispersal events in Thelymitra highlights the importance of the prevailing westerly winds in the Southern Hemisphere for the present-day distribution of the genus, giving rise to the Thelymitra floras of Tasmania, New Zealand and New Caledonia, which were inferred to be of comparatively recent origin.