Mating-system evolution in Bulbophyllum (Orchidaceae)

The orchid family (Orchidaceae) is characterized by some of the most sophisticated cross-pollination mechanisms known, but relatively little consideration has been given to the fact that auto-pollination (i.e. self-pollination/selfing without the aid of pollinators) is widespread in the family. Moreover, especially in tropical orchids, very limited data exist on the mating-system evolution in general, and the evolutionary pathway under which auto-pollination occurs in particular. By adopting both macro- and micro-evolutionary approaches, the major aim of this proposal is to reconstruct shifts in orchid mating-system evolution in a tropical biome, using members of the genus Bulbophyllum THOUARS (Orchidaceae/Epidendroidea) from the Southwest Indian Ocean region as a suitable model system, as they include both outcrossing and auto-pollinating species. The macro-evolutionary approach first will focus on refined molecular-phylogenetic (ITS, cpDNA, single/low-copy nuclear genes), morphological and pollination-experimental studies in the previously identified Bulbophyllum "clade C" comprising c. 26 species, mainly distributed in Madagascar, but also occurring in the remote oceanic islands of La Réunion/Mauritius (Mascarenes) and the Comores, and on the East African continent. These analyses will then be complemented by reconstructions (optimizations) of ancestral reproductive/floral-characters, areas and habitat preferences in conjunction with a molecular dating approach using paleogeological and fossil-based calibrations. Together, this will allow us to infer how mating types (outcrossing vs. auto-pollinated), and associated floral traits, have changed within clade C over evolutionary time and under which spatial, temporal and ecological circumstances. In addition, the proposed micro-evolutionary studies will focus on a clade C sister-species pair, B. bicoloratum SCHLTR. and B. occultum THOUARS, both with supposedly outcrossing populations in Madagascar ("the mainland"), but with exclusively auto-pollinating populations of B. occultum also occurring in La Réunion, Mauritius, and the Comores. Here, we will employ population genetic surveys of cpDNA haplotype sequence and nuclear microsatellite variation at both the intra- and interspecific level, complemented by detailed floral-trait measurements and pollination experiments, to determine the reproductive, floral-morphological and population genetic consequences associated with remote oceanic island colonization of B. occultum.

Background The shift from outcrossing to selfing is one the most common evolutionary changes in flowering plants. Selfing in orchids is typically assumed to provide reproductive assurance under conditions of pollinator and/or mate limitation, especially at northern latitudes. However, in tropical orchids, little is known about the frequency and evolutionary consequences of shifts in mating system. Aims and Methods We investigated shifts from outcrossing to selfing (auto-pollination) over evolutionary and ecological time scales in a monophyletic group of epiphytic, self-compatible Bulbophyllum orchids (clade C) from Madagascar and La Réunion (c. 30 spp.). We addressed this issue by conducting pollination experiments and floral micro-morphological studies, accompanied by molecular phylogenetic approaches at the clade level (e.g., molecular dating, ancestral character reconstructions) and population genetic (DNA fingerprint, chloroplast DNA sequences, and nuclear microsatellite) surveys in a sister-species pair from Madagascar (B. bicoloratum/B. occultum) and La Réunion (only B. occultum). Key Results Our studies revealed that mating type is directly related to floral structure, most often involving the presence or absence of a physical barrier termed rostellum, which usually separates the pollinia from the stigmatic cavity below. Specifically, we identified eight clade C species, in which auto-pollinating morphs (selfers), either lacking a rostellum or (rarely) possessing a stigmatic rostellum, co-exist with their pollinator-dependent conspecifics (outcrossers). Phylogenetic analyses further revealed that rostellum presence (viz. outcrossing) is the ancestral state of the entire clade, which diversified at a constant rate from the Late Tertiary onwards (? 5.3 Ma). However, transition to selfing has apparently occurred only since the Late Pleistocene (? 1.0 Ma) and within multiple unrelated species. Moreover, remote island populations of B. occultum in La Réunion were found to be fixed for the selfing morph and genetically impoverished, whereas those from Madagascar varied in mating type but also showed genetic signs of range fragmentation (insularization) and inbreeding. Conclusions Our results suggest that Bulbophyllum species have an intrinsic genetical and developmental lability toward auto-pollination via rostellum abortion, allowing fast evolutionary response under environmental conditions favouring reproductive assurance. The most likely catalyst initiating the evolution of increased selfing in multiple clade species is demographic bottlenecks associated with recent habitat fragmentation due to human-mediated forest destruction in Madagascar. Hence, the parallel and recent evolution of selfing in these tropical orchids is probably not a Dead End, but rather a means avoiding extinction in the face of irresponsible over-exploitation of natural resources by humans in one of the worlds major biodiversity hotspots.