Many species are poorly known, with the sum of our knowledge represented by specimens in museums. For assessment of conservation status the most enigmatic and challenging species are probably those known only from a single specimen. We examine the potential persistence of such species using the orchid flora of Madagascar as a case study. We apply a statistical method that tests the likelihood of species presence in relation to the time when a species was collected and a measure of annual collection effort, calculated in three ways based on specimen collection over time. The results suggest that as of 2000 up to nine of the 236 orchid species known from a single specimen may be inferred to be extinct under at least one of the three methods of estimating collection effort and extinction. In addition, up to two additional species are likely to be extinct by 2018 assuming no new collections were made by that time. Substantial collection effort and/or additional evidence will be needed to reach a decision on the persistence of more recently observed species known only from a single collection. This represents a challenge for conservation practitioners.

This study examines the microtubular cytoskeleton during megasporogenesis in the Nun orchid, Phaius tankervilliae. The subepidermal cell located at the terminal end of the nucellar filament differentiates first into an archesporial cell and then enlarges to become the megasporocyte. The megasporocyte undergoes the first meiotic division, giving rise to two dyad cells of unequal size. Immunostaining reveals that microtubules become more abundant as the megasporocyte increases in size. Microtubules congregate around the nucleus forming a distinct perinuclear array and many microtubules radiate directly from the nuclear envelope. In the megasporocyte, prominent microtubules are readily detected at the chalazal end of the cell cytoplasm. After meiosis I, the chalazal dyad cell expands in size at the expense of the micropylar dyad cell. At this stage, new microtubule organizing centres can be found at the corners of the cells. The appearance of these structures is stage-specific and they are not found at any other stages of megasporogenesis. The functional dyad cell undergoes the second meiotic division, resulting in the formation of two megaspores of unequal size. The chalazal megaspore enlarges and eventually gives rise to the embryo sac. As the functional megaspore expands, the microtubules again form a distinct perinuclear array with many microtubules radiating from the nuclear envelope. A defined cortical array of microtubules has not been found in P. tankervilliae during the course of megasporogenesis.

The processes of symbiotic germination and seedling development were analysed in the myco-heterotrophic orchid Corallorhiza trifida, seeds of which were buried in ‘packets’ either adjacent to or at varying distances from adult plants in defined communities of ectomycorrhizal tree species. Germination occurred within eight months of burial under Betula–Alnus and within seven months under Salix repens. It was always associated with penetration of the suspensor by a clamp-forming mycorrhizal fungus. Four distinct developmental stages were defined and the rates of transition through these stages were plotted. There was no evidence of a relationship between extent of germination or rate of development and the presence of naturally distributed plants of C. trifida at the spatial scale of 1 m. The best germination and the most rapid rate of development of C. trifida seedlings occurred in a Salix repens community located at a considerable distance from any extant C. trifida population. Determination of internal transcribed spacer (ITS) RFLPs and of gene sequences of the fungi involved in symbiotic germination and growth of C. trifida, revealed them to belong exclusively to the Thelephora–Tomentella complex of the Thelephoraceae. These fungi are known also to be ectomycorrhizal associates of trees. It is hypothesized that the rate of growth of the C. trifida seedlings is determined by the ability of the fungal symbionts to transfer carbon from their ectomycorrhizal co-associates.

Seeds of the orchid species, Spiranthes sinensis (Pers.) Ames, were sterilized and germinated in vitro with the symbiotic fungus Ceratobasidium cornigerum (Bourdot) Rogers. Colonized embryos developed into protocorms and these were examined for changes in microtubule arrays, after initial invasion of fungal hyphae into embryos and during peloton formation and degradation. Methods utilized to detect microtubules included immunofluorescence combined with laser scanning confocal microscopy, conventional transmission electron microscopy combined with morphometric analysis, and immunogold labelling. Microtubules were regularly found in close association with intracellular hyphae and degraded hyphal masses. Cortical microtubules disappear during peloton formation but reappear in cells that show fungal lysis. With conventional transmission electron microscopy and immunogold labelling the microtubules associated with fungal hyphae and degenerated hyphal masses were located close to the perifungal membrane that separates fungal hyphae from protocorm cytoplasm.

Little is known about non-mycorrhizal endophytic fungi in tropical orchids; still less is known about how endophytes vary within and between individual orchid plants. Fungal endophytes were isolated from roots and leaves of epiphytic and lithophytic orchids in the genus Lepanthes; seven species, from rainforests in Puerto Rico, were sampled. The endophytes observed most frequently were Xylaria species and Rhizoctonia-like fungi, found in 29% of roots and 19% of leaves, and 45% of roots and 31% of leaves, respectively. Five deuteromycete genera were also isolated, occurring in 19% of roots and 43% of leaves (combined). At least nine species of Xylaria were found, with several species sometimes occurring in a single plant. Differences between roots and leaves in frequency of Xylaria and Rhizoctonia isolates were not significant, although differences among orchid species in number and types of endophytes were. Heterogeneity of endophytes in single plants and plant organs was greater than differences between species. Many Lepanthes species are very restricted in distribution, and knowledge of their interactions with endophytes might be useful in species management.