Tulasnella spp. as saprotrophic and mycorrhizal fungi of tropical orchids: morphology, molecular taxonomy, and ecology

Tulasnella species (Tulasnellaceae, Cantharellales, Basidiomycota) form inconspicuous basidiomata on rotten branches or trunks of trees, difficult to find and recognize in nature. However, according to ultrastrucural and molecular data, species of Tulasnellaceae are the most frequent mycorrhriza forming fungi (mycobionts) of green, photosynthetic orchids worldwide. Species of Tulasnellaceae were also found as prominent mycobionts of the extraordinary diverse orchids in tropical montane rainforest of Southern Ecuador. Orchids obligately depend on mycobionts during the juvenile stage when the fungi have to deliver carbon to the non-photosynthetic protocorm and thus the fungi substantially influence the establishment of orchids in the wild. Species of Tulasnellaceae can acquire carbon from decaying bark or wood by specific saprotrophic capabilities as was recently proven through comparative genomics that included data on decay enzymes from Tulasnella cf. calospora isolated from orchid mycorrhizae (Anacamptis laxiflora, Italy). Thus, species of Tulasnellaceae can be saprotrophs and symbionts simultaneously. It is currently under discussion, whether specific species of Tulasnella are required for seed germination and establishment of distinct terrestrial and epiphytic orchids in nature or if species of Tulasnella are generalists concerning their association with orchids. The inconsistences in species concepts and taxonomy of Tulasnella spp., however, strongly impede progress in this field of research. The aim of the present study was, therefore, to revise the species concepts by combining, for the first time, morphological and molecular data from basidiomata. Specimens were collected in tropical Andean forest in Southern Ecuador and in temperate forests in Germany. Additional specimens were loaned from fungaria. In total, 205 specimens, corresponding to 16 own samples and 189 specimens from fungaria were analyzed. The mycobiont relationships of Tulasnella spp. with orchids from the sampling area in Ecuador were studied in populations of Epidendrum rhopalostele. The basis for molecular-phylogenetic analysis was completed by data obtained from own previous investigations on mycobionts from the investigation area and Tulasnella isolates from Australia. 30 morphospecies are illustrated and delimited by a morphological key based on traditional species concepts. Tulasnella andina from Ecuador and Tulasnella kirschneri from China are presented as species new to science. Tulasnella cruciata is described from herbarium material for the first time. Tulasnella aff. eichleriana and T. violea are reported for the first time from Ecuador. Molecular sequences of two Tulasnella spp. isolated from mycobionts of Epidendrum rhopalostele cannot be related to any morphological species concept. Statistical analyses suggest that conventional diagnostic using morphological characteristics is ambiguous for delimiting morphologically similar species. For the first time sequences of the ITS-5.8S rDNA region were obtained after cloning from fresh basidiomata. Extraction of DNA from herbarium specimens was, however, unsuccessful. Sequences from 16 fresh basidiomata, six pure cultures, and sequences of orchids mycorrhizae (e.g. from Epidendrum rhopalostele) available in the database GenBank were analyzed. Proportional variability of ITS-5.8S rDNA sequences within and among cultures and within and among specimens were used to designate morphospecies. Results suggest an intragenomic variation of less than 2 %, an intraspecific variation of up to 4 % and an interspecific divergence of more than 9 % for Tulasnella spp. Four percent of intraspecific divergence was defined as a minimum threshold for delimiting phylogenetic species. This threshold corroborates the so far used 3 % to 5 % divergence in delimitation of operational taxonomic units of Tulasnella mycobionts. Quite a number of sequences of Tulasnella are available in GenBank, mostly obtained from direct PCR amplification from orchid mycorrhizae. By including closely related sequences in the phylogenetic analysis, several morphological cryptic species of Tulasnella, mostly from Ecuador, were found. Arguments are given for molecular support of the new species Tulasnella andina and the established species Tulasnella albida, T. asymmetrica, T. eichleriana, T. tomaculum, and T. violea. Thus, by combining molecular and morphological data species concepts in Tulasnella are improved. The definitions of Tulasnella calospora and T. deliquescens, however, remain phylogenetically inconsistent. The present investigation is a first step to expand our knowledge on the intraand interspecific morphological and molecular variability of Tulasnella spp. and to delimit species relevant for studies on ecology and communities of orchids and Tulasnellaceae.Die Familie der Tulasnellaceae gehört zur Ordnung der Cantharellales, welche eine basale Stellung innerhalb der Agaricomycetes (Basidiomycota) einnimmt. Die Tulasnellaceae bestehen aus drei Gattungen: Pseudotulasnella, Stilbotulasnella und Tulasnella. Die Gattung Pseudotulasnella mit der Art Pseudotulasnella guatemalensis sowie die Gattung Stilbotulasnella mit der Art Stilbotulasnella conidiophora sind monotypisch. Die Gattung Tulasnella ist die diverseste Gattung mit vielen Arten innerhalb dieser Familie. Tulasnella spp. sind hauptsächlich charakterisiert durch fortlaufend septierte Hyphen, die in manchen Arten Schnallen tragen. Die Basidien sind nicht septiert, generell kugelförmig oder subglobos oder keulenförmig bis obclavat. Cystiden, wenn vorhanden, sind Gloeocystiden. Die Sterigmen sind kugelförmig, ellipsoid oder spindelförmig. Manche Arten bilden auch Chlamydosporen. Die Basidiosporen sind glatt und können unterschiedliche Formen haben, wie z.B. kugelförmig, subglobos, länglich, elliptisch, bohnenförmig, allantoid, spiralig, sigmoid oder bauchig. Sie produzieren laufend sekundäre Basidiosporen. Weiterhin bilden Tulasnella spp. unscheinbare Basidiomata auf verrottenden Ästen oder Baumstämmen, welche in der Natur schwierig zu entdecken sind. ... Die Ergebnisse deuten auf eine intragenomische Variabilität von weniger als 2%, eine intraspezifische Variabilität von bis zu 4% und eine interspezifische Divergenz von mehr als 9% für Tulasnella spp. hin. Vier Prozent Divergenz wurde als Minimum-Schwellenwert für die Abgrenzung der phylogenetischen Arten definiert. Dieser Schwellenwert bestätigt die bis jetzt genutzte 3% bis 5% Divergenz bei der Abgrenzung von praktikablen taxonomischen Einheiten von Tulasnella-Mykobionten oder für das Abschätzen der Diversität in anderen Pilzgruppen