Temporal variation in mycorrhizal diversity and carbon and nitrogen stable isotope abundance in the wintergreen meadow orchid Anacamptis morio.

Many adult orchids, especially photoautotrophic species, associate with a diverse range of mycorrhizal fungi, but little is known about the temporal changes that might occur in the diversity and functioning of orchid mycorrhiza during vegetative and reproductive plant growth. Temporal variations in the spectrum of mycorrhizal fungi and in stable isotope natural abundance were investigated in adult plants of Anacamptis morio, a wintergreen meadow orchid. Anacamptis morio associated with mycorrhizal fungi belonging to Tulasnella, Ceratobasidium and a clade of Pezizaceae (Ascomycetes). When a complete growing season was investigated, multivariate analyses indicated significant differences in the mycorrhizal fungal community. Among fungi identified from manually isolated pelotons, Tulasnella was more common in autumn and winter, the pezizacean clade was very frequent in spring, and Ceratobasidium was more frequent in summer. By contrast, relatively small variations were found in carbon (C) and nitrogen (N) stable isotope natural abundance, A. morio samples showing similar (15)N enrichment and (13)C depletion at the different sampling times. These observations suggest that, irrespective of differences in the seasonal environmental conditions, the plant phenological stages and the associated fungi, the isotopic content in mycorrhizal A. morio remains fairly constant over time.

Gene expression in mycorrhizal orchid protocorms suggests a friendly plant-fungus relationship.

Orchids fully depend on symbiotic interactions with specific soil fungi for seed germination and early development. Germinated seeds give rise to a protocorm, a heterotrophic organ that acquires nutrients, including organic carbon, from the mycorrhizal partner. It has long been debated if this interaction is mutualistic or antagonistic. To investigate the molecular bases of the orchid response to mycorrhizal invasion, we developed a symbiotic in vitro system between Serapias vomeracea, a Mediterranean green meadow orchid, and the rhizoctonia-like fungus Tulasnella calospora. 454 pyrosequencing was used to generate an inventory of plant and fungal genes expressed in mycorrhizal protocorms, and plant genes could be reliably identified with a customized bioinformatic pipeline. A small panel of plant genes was selected and expression was assessed by real-time quantitative PCR in mycorrhizal and non-mycorrhizal protocorm tissues. Among these genes were some markers of mutualistic (e.g. nodulins) as well as antagonistic (e.g. pathogenesis-related and wound/stress-induced) genes. None of the pathogenesis or wound/stress-related genes were significantly up-regulated in mycorrhizal tissues, suggesting that fungal colonization does not trigger strong plant defence responses. In addition, the highest expression fold change in mycorrhizal tissues was found for a nodulin-like gene similar to the plastocyanin domain-containing ENOD55. Another nodulin-like gene significantly more expressed in the symbiotic tissues of mycorrhizal protocorms was similar to a sugar transporter of the SWEET family. Two genes coding for mannose-binding lectins were significantly up-regulated in the presence of the mycorrhizal fungus, but their role in the symbiosis is unclear.

A new species of Hoya R.Br. (Apocynaceae, Asclepiadoideae) from the Philippines.

Hoyamedusa M.D.De Leon, Cabactulan, Cuerdo & Rodda, sp. nov. (Apocynaceae, Asclepiadoideae) is described from the Philippines. Even though numerous taxa with a shrubby habit from this area are known, it can be immediately separated because of its urceolate corolla and prominent elongated corona lobes. No other species in the genus possesses such a combination of characters.

Photosynthetic Mediterranean meadow orchids feature partial mycoheterotrophy and specific mycorrhizal associations.

PREMISE OF THE STUDY: We investigated whether four widespread, photosynthetic Mediterranean meadow orchids (Ophrys fuciflora, Anacamptis laxiflora, Orchis purpurea, and Serapias vomeracea) had either nutritional dependency on mycobionts or mycorrhizal fungal specificity. Nonphotosynthetic orchids generally engage in highly specific interactions with fungal symbionts that provide them with organic carbon. By contrast, fully photosynthetic orchids in sunny, meadow habitats have been considered to lack mycorrhizal specificity. METHODS: We performed both culture-dependent and culture-independent ITS sequence analysis to identify fungi from orchid roots. By analyzing stable isotope ((13)C and (15)N) natural abundances, we also determined the degree of autotrophy and mycoheterotrophy in the four orchid species. KEY RESULTS: Phylogenetic and multivariate comparisons indicated that Or. purpurea and Oph. fuciflora featured lower fungal diversity and more specific mycobiont spectra than A. laxiflora and S. vomeracea. All orchid species were significantly enriched in (15)N compared with neighboring non-orchid plants. Orchis purpurea had the most pronounced N gain from fungi and differed from the other orchids in also obtaining C from fungi. CONCLUSIONS: These results indicated that even in sunny Mediterranean meadows, orchids may be mycoheterotrophic, with correlated mycorrhizal fungal specificity.

Pectin localization in the Mediterranean orchid Limodorum abortivum reveals modulation of the plant interface in response to different mycorrhizal fungi.

In most mycorrhizal symbioses, phylogenetically distinct fungi colonize simultaneously the roots of individual host plants. A matter of debate is whether plants can distinguish among these fungal partners and differentiate their cellular responses. We have addressed this question in the orchid mycorrhizal symbiosis, where individual roots of the Mediterranean species Limodorum abortivum can be colonized by a dominant unculturable fungal symbiont belonging to the genus Russula and by more sporadic mycelia in the genus Ceratobasidium (form-genus Rhizoctonia). The phylogenetic position of the Ceratobasidium symbionts was further investigated in this work. Both Russula and Ceratobasidium symbionts form intracellular coils in the cortical roots of L. abortivum, but hyphae are very different in size and morphology, making the two fungi easily distinguishable. We have used John Innes Monoclonal 5, a widely used monoclonal antibody against pectin, to investigate the composition of the symbiotic plant interface around the intracellular coils formed by the two fungal partners. Immunolabelling experiments showed that pectin is exclusively found in the interface formed around the Ceratobasidium, and not around the Russula symbiont. These data indicate that the plant responses towards distinct mycorrhizal fungal partners can vary at a cellular level.

Plastome evolution and organisation in the Hoya group (Apocynaceae).

The genus Hoya is highly diverse and many of its species are popular ornamental plants. However, the relationships between Hoya and related genera (the Hoya group) are not fully resolved. In this study, we report 20 newly sequenced plastomes of species in the Hoya group. The complete plastomes vary in length from 175,405 to 178,525 bp while the LSCs vary from 90,248 to 92,364 bp and the complete SSCs vary from 2,285 to 2,304 bp, making the SSC in the Hoya group one of the shortest known in the angiosperms. The plastome structure in the Hoya group is characterised by a massive increase in the size of the inverted repeats as compared to the outgroups. In all ingroup species, the IR/SSC boundary moved from ycf1 to ndhF while this was not observed in outgroup taxa, making it a synapomorphy for the Hoya group. We have also assembled the mitogenome of Hoya lithophytica, which, at 718,734 bp, is the longest reported in the family. The phylogenetic analysis using exons from 42 taxa in the Hoya group and three outgoups confirms that the earliest divergent genus in the Hoya group is Papuahoya, followed by Dischidia. The relationship between Dischidia and the clade which includes all Hoya and Oreosparte taxa, is not fully supported. Oreosparte is nested in Hoya making it paraphyletic unless Clemensiella is recognised as a separate genus.

Heterostemma cucphuongense (Apocynaceae, Asclepiadoideae), a new species from Vietnam.

Heterostemma cucphuongense (Apocynaceae, Asclepiadoideae), a new species from Vietnam is described, illustrated and compared with the similar species Heterostemma succosum Kerr. Heterostemma cucphuongense differs from H. succosum by the morphology of the rachis of the inflorescence, the margins of the corolla lobes and the colour of the adaxial surface of the corolla.

Hoya of the Philippines part I. Hoya migueldavidii (Apocynaceae, Asclepiadoideae), a new species from Northern Mindanao, Philippines.

A new species of Hoya R.Br. from Mindanao (Philippines), Hoya migueldavidii Cabactulan, Rodda & Pimentel, is described and illustrated. It is a member of Hoya section Acanthostemma (Blume) Kloppenb. that is particularly speciose in the Philippines. It is compared with the similar Hoya loheri Kloppenb, also endemic of the Philippines, from which it differs in indumentum of the vegetative parts (pubescent vs. glabrous), the shape of the corolla (almost spherical vs. partly flattened) and the type of gynostegium (not stipitate vs. stipitate).

Hoya isabelchanae Rodda & Simonsson, a new, showy species of Hoya R.Br. (Apocynaceae, Asclepiadoideae) with pomegranate red flowers from Sulawesi, Indonesia.

A new species of Hoya R.Br. from Sulawesi (Indonesia), Hoya isabelchanae Rodda & Simonsson, is described and illustrated. It is one of the largest flowered species in Hoya section Acanthostemma (Blume) Kloppenb. Its flowers are of comparable size to those of Hoya benchaii Gavrus et al., Hoya kloppenburgii T.Green, Hoya rundumensis (T.Green) Rodda & Simonsson and Hoya sigillatis T.Green ssp. sigillatis, all from Borneo. Among Sulawesi species it is compared with the vegetatively similar Hoya brevialata Kleijn & van Donkelaar and Hoya pallilimba Kleijn & van Donkelaar.

Jasminanthes xuanlienensis (Apocynaceae, Asclepiadoideae), a new species from Vietnam.

Jasminanthes xuanlienensis (Apocynaceae, Asclepiadoideae), a new species from Vietnam is described, illustrated and compared with its five congeners. Jasminanthes xuanlienensis differs distinctly from congeners by the longer peduncles (14-18 cm vs. 4 cm at most in Jasminanthes pilosa and Jasminanthes saxatilis, salmon-pink color of the inner corolla lobes (white or greenish in the other species), and corolla tube length (12.0-14.5 mm vs. shorter or longer in congeners).

Two new species of Hoya R.Br. (Apocynaceae, Asclepiadoideae) from Borneo.

Two new Hoya R.Br. species from Borneo are described and illustrated. The first, Hoyaruthiae Rodda was collected in Sabah on Bukit Baturong, a limestone outcrop. It is one of the few species in the genus to have clear exudate. It is compared with the morphologically related Hoyauncinata Teijsm. and Binn. The other, Hoyabakoensis Rodda, was collected in the kerangas forests of Bako National Park. It belongs to HoyasectionAcanthostemma (Bl.) Kloppenb., a section with numerous members in the Philippines but under-represented in Borneo.

Cryopreservation of orchid mycorrhizal fungi: a tool for the conservation of endangered species.

The effectiveness of cryopreservation at --80 °C on orchid mycorrhizal fungi was assessed by testing the symbiotic ability of ten fungal isolates following cryo-storage for 10-24 months. The results obtained prove the efficacy of the method, thus providing a valuable tool for ex situ conservation.

C and N stable isotope signatures reveal constraints to nutritional modes in orchids from the Mediterranean and Macaronesia.

We compared the nutritional modes and habitats of orchids (e.g., autotrophic, partially or fully mycoheterotrophic) of the Mediterranean region and adjacent islands of Macaronesia. We hypothesized that ecological factors (e.g., relative light availability, surrounding vegetation) determine the nutritional modes of orchids and thus impose restrictions upon orchid distribution. Covering habitats from dark forests to open sites, orchid samples of 35 species from 14 genera were collected from 20 locations in the Mediterranean and Macaronesia to test for mycoheterotrophy. Mycorrhizal fungi were identified via molecular analyses, and stable isotope analyses were applied to test whether organic nutrients are gained from the fungal associates. Our results show that orchids with partial or full mycoheterotrophy among the investigated species are found exclusively in Neottieae thriving in light-limited forests. Neottioid orchids are missing in Macaronesia, possibly because mycoheterotrophy is constrained by the lack of suitable ectomycorrhizal fungi. Furthermore, most adult orchids of open habitats in the Mediterranean and Macaronesia show weak or no N gains from fungi and no C gain through mycoheterotrophy. Instead isotope signatures of some of these species indicate net plant-to-fungus C transfer.