Abstract:Orchid flora of Madagascar is one of most diverse with nearly 1,000 orchid taxa of which about 90% are endemic to this biodiversity hotspot. The Itremo Massif in the Central Highlands of Madagascar with a Highland Subtropical climate range encompasses montane grassland, igneous and metamorphic rock outcrops, and gallery and tapia forests. Our study focused on identifying culturable mycorrhizae from epiphytic, lithophytic and terrestrial orchid taxa to understand their diversity and density in a spatial matrix that is within the protected areas.We have collected both juvenile and mature roots from 40 orchid taxa for isolating the orchid mycorrhizal fungi (OMF), to culture, identify and store in liquid nitrogen for future studies. Twelve operational taxonomic units (OTUs), of three known orchid mycorrhizal genera, were recognized by analysis of internal transcribed spacer (ITS) sequences of 86 isolates and, by comparing with GenBank database entries, each OTU was shown to have closely related fungi that were also found as orchid associates. Orchid and fungal diversity were greater in gallery forests and open grasslands which is very significant for future studies and orchid conservation. As far as we know this is the first ever report of detailed identification of mycorrhizae from Powered by Editorial Manager® and ProduXion Manager® from Aries Systems CorporationMadagascar. This study will help start to develop a programme for identifying fungal symbionts from this unique biodiversity hotspot which is undergoing rapid ecosystem damage and species loss. The diversity of culturable mycorrhizae, density and their distribution within the Itremo orchid hotspot areas will be discussed. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation

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ProtocormS of an EPiPhytic orchid (EpidEndrum amphistomum a. richard) rEcovErEd in situ, and SubSEquEnt idEntification of aSSociatEd mycorrhizal fungi uSing molEcular markErS L aw r ence w. Z et t L er 1, * , L au r a L . cor ey 1 , L a r ry w. r ich a r dson 2 , a pr i L y. ros s 1, 3 , and L i L L i a n MoL L e r-Jacob s 1, 4 1 department of biology, illinois college, 1101 west college avenue, Jacksonville, iL 62650 usa 2 Florida panther national wildlife refuge, u.s. Fish and wildlife service, 3860 tollgate boulevard, suite 300, naples, FL 34114, usa 3 current address: department of public heath, university of illinois at springfield, springfield iL, 62703 usa 4 current address: department of entomology, 501 asi building, pennsylvania state university, university park, pa 16802 usa * corresponding author: lwzettle@ic.edu; tel.: (217) 245-3479; Fax: (217) 245-3358 AbstrActEpiphytic orchids have received considerable study, yet little has been published on their germination requirements in situ involving mycorrhizal fungi. Such research has been hampered by the small, dust-like size of seeds and leafless seedlings (protocorms) which are difficult to pinpoint on natural substrates, especially those on arboreal substrates (tree limbs). We report a novel seed sowing and retrieval method, modified from one applied to terrestrial orchids, used in the acquisition of epiphytic orchid protocorms from the Florida Panther National Wildlife Refuge. Seeds from two epiphytic orchid species (Epidendrum amphistomum A. Richard, E. nocturnum Jacquin) were placed in separate nylon mesh packets secured within 35 mm plastic slide mounts, and affixed to tree bark using gutter mesh and a staple gun. To confirm that the embryos were viable, some seeds were also sown on asymbiotic media in the laboratory which subsequently germinated after 52 days incubation. Of 60 packets distributed among 18 tree limb sites, one packet -harboring seeds of E. amphistomum affixed to pop ash (Fraxinus caroliniana Mill.) on a moss substrate -harbored protocorms after 267 days. Using molecular markers, a fungus assignable to the Ceratobasidiaceae, appears to be the mycorrhizal associate of these protocorms suggesting that this fungus may be associated with the germination process in situ.

Las relaciones micorríticas obligadas de las orquídeas epífitas – un componente clave en el modelo integrado de conservación – siguen desconocidas. Los estudios existentes han revelado que estas plantas, así como sus equivalentes terrestres, se asocian normalmente con basidiomicetos ubícuitos (e.g. Tulasnellaceae); sin embargo pocos estudios han verificado su papel fisiológico. Dos cepas del hongo micorrítico (UAMH 11541, UAMH 11543) fueron aisladas de las raíces de una especie de orquídea epifítica del sur de Florida, Encyclia tampensis – una obtenida de una plántula joven y la otra obtenida de una planta madura. Las semillas de cuatro taxones epífitos fueron inoculadas por separado con los dos hongos aislados in vitro: E. tampensis, Epidendrum amphistomum, Epidendrum nocturnum y Prosthechea cochleata. Más de un tercio de las semillas de E. tampensis y E. nocturnum (ambos inoculados) desarrollaron hojas cuando fueron mantenidos en total obscuridad por más de cien días. No se detectaron diferencias significativas entre las dos cepas del hongo con respecto a la germinación y la interacción entre el hongo y las semillas procedentes de diferentes fuentes (ANOVA, α= 0.05). Usando amplificación y secuenciación ITS, ambas cepas fueron identificadas como teleomorfos, Tulasnella irregularis (Basidiomycota: Tulasnellaceae) y ambas resultaron genéticamente idénticas con un elevado grado (98%) de seguridad. De esta manera, tanto la germinación simbiótica así como los resultados de la secuenciación ITS concuerdan en identificar con certeza ambas cepas como el mismo hongo. Este artículo trata de elucidar las interacciones entre orquídeas epífitas y hongos micorríticos y también subraya la necesidad de identificar, comprobar (a través de germinación simbiótica), y preservar los hongos micorríticos necesarios para fines de conservación.

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