Cryptic species revealed by molecular phylogenetic analysis of sequences obtained from basidiomata of Tulasnella

Abstract

Delimitation of species and the search for a proper threshold for defining phylogenetic species in fungi are under discussion. In this study, morphological and molecular data are correlated to delimit species of Tulasnella, the most important mycobionts of Orchidaceae, which suffer from poor taxonomy. Resupinate basidiomata of Tulasnella species were collected in Ecuador and Germany, and 11 specimens (seven from Ecuador, four from Germany) were assigned to traditional species concepts by use of morphological keys. The specimens were compared by micro-anatomical examination with 75 specimens of Tulasnella borrowed from fungaria to obtain better insights on variation of characters. Sequences of the ITS region (127) were obtained after cloning from the fresh basidiomata and from pure cultures. Proportional variability of ITS sequences was analyzed within and among the cultures and the specimens designated to different morphospecies. Results suggested an intragenomic variation of less than 2%, an intraspecific variation of up to 4% and an interspecific divergence of more than 9% in Tulasnella. Cryptic species in Tulasnella, mostly from Ecuador, were revealed by phylogenetic analyses with 4% intraspecific divergence as a minimum threshold for delimiting species. Conventional diagnostic morphological characters appeared insufficient for species characterization. Arguments are presented for molecular delimitation of the established species Tulasnella albida, T. asymmetrica, T. eichleriana, T. cf. pinicola, T. tomaculum and T. violea.

• interspecific divergence
• intragenomic variability
• intraspecific variability
• ITS-5.8S region
• morphospecies
• teleomorphic states

Acknowledgments

The authors thank Martin Bidartondo for providing six sequences of Tulasnella included in the phylogenetic analysis. We also appreciate the help by Roland Kirschner who let us revise his personal collection of Tulasnella and Franz Oberwinkler who provided samples of Tulasnella collected by him, deposited at the Eberhard-Karls University of Tübingen, Germany. We are grateful for the help of Andreas Beck, Ralph Mangelsdorff for critical comments on the manuscript and Jascha Weisenborn for help and assistance in the laboratory in Germany. The research was generously supported by Deutsche Forschungsgemeinschaft (DFG RU 816) and German Academic Exchange Service (DAAD). Nature and Culture International (NCI) provided research facilities at RBSF.