Members of Sebacinales subgroup B form mycorrhizae with epiphytic orchids in a neotropical mountain rain forest

Previous investigations revealed that epiphytic orchids in a mountain rain forest in southern Ecuador formed mycorrhizae with diverse members of Tulasnellales. Using specific primers, we now show that the same orchids are also associated with Sebacinales. Ultrastructural observations confirmed the Sebacinales mycobionts in situ. Mycorrhizae of flowering individuals of Stelis hallii, S. superbiens, S. concinna and Pleurothallis lilijae were sampled in different forest types of the mountain rain forest of southern Ecuador along an altitudinal gradient between 1,850 and 2,100 m a.s.l. Phylogenetic analysis of fungal nuclear rDNA sequences coding for the ribosomal large subunit (nucLSU) showed the presence of eight sequence types based on proportional differences of < 1% bp. All sequence types clustered in the Sebacinales subgroup B which also contained sequences of mycobionts from ericads and terrestrial orchids. Sequences of the nuclear rDNA 5.8S subunit, including parts of the internal transcribed spacers ITS1 and ITS2 (5.8-ITS) from the mycobionts of the epiphytic orchids, were distinct from published sequences of sebacinoid mycobionts of green terrestrial orchids and ericads. Sebacinales sequences from different epiphytic orchid species differed at least by 1% bp as was previously found for Tulasnella sequences. Sebacinales occurred less frequently and with a lower number of sequence types than Tulasnellales, but distribution along the altitudinal gradient was similar.