Mycol. Res. 108 (9): 1003–1010 (September 2004). f The British Mycological Society 1003 DOI: 10.1017/S0953756204000772 Printed in the United Kingdom. Sebacinales : a hitherto overlooked cosm of heterobasidiomycetes with a broad mycorrhizal potential* Michael WEISS1**, Marc-André SELOSSE2, Karl-Heinz REXER3, Alexander URBAN4 and Franz OBERWINKLER1 1 Botanisches Institut, Universität Tübingen, Auf der Morgenstelle 1, D-72076 Tübingen, Germany. UMR CNRS 7138, Syste´matique, Adaptation et Evolution, Muse´um d’Histoire Naturelle, 43 rue Cuvier, F-75005 Paris, France. 3 Fachbereich Biologie, Universität Marburg, Karl-von Frisch-Straße 1, D-35032 Marburg, Germany. 4 Institut für Botanik, Universität Wien, Rennweg 14, A-1030 Wien, Austria. E-mail : michael.weiss@uni-tuebingen.de 2 Received 28 April 2004; accepted 16 June 2004. Within the basidiomycetes, the vast majority of known mycorrhizal species are homobasidiomycetes. It was therefore surprising when molecular and ultrastructural studies revealed a broad diversity of mycorrhizal associations involving members of the heterobasidiomycetous Sebacinaceae, fungi which, due to their inconspicuous basidiomes, have been often overlooked. To investigate the phylogenetic position of the Sebacinaceae within the basidiomycetes and to infer phylogenetic relationships within the Sebacinaceae, we made molecular phylogenetic analyses based on nuclear rDNA. We present a well-resolved phylogeny of the main lineages of basidiomycetes which suggests that the Sebacinaceae is the most basal group with known mycorrhizal members. Since more basal taxa of basidiomycetes consist of predominantly mycoparasitic and phytoparasitic fungi, it seems possible that a mycorrhizal life strategy, which was transformed into a saprotrophic strategy several times convergently, is an apomorphic character for the Hymenomycetidae. Mycorrhizal taxa of Sebacinaceae, including mycobionts of ectomycorrhizas, orchid mycorrhizas, ericoid mycorrhizas, and jungermannioid mycorrhizas, are distributed over two subgroups. One group contains species with macroscopically visible basidiomes, whereas members of the other group probably lack basidiomes. Sebacina appears to be polyphyletic; current species concepts in Sebacinaceae are questionable. Sebacina vermifera sensu Warcup & Talbot consists of a broad complex of species possibly including mycobionts of jungermannioid and ericoid mycorrhizas. This wide spectrum of mycorrhizal types in one fungal family is unique. Extrapolating from the known rDNA sequences in Sebacinaceae, it is evident that there is a cosm of mycorrhizal biodiversity yet to be discovered in this group. Taxonomically, we recognise the Sebacinaceae as constituting a new order, the Sebacinales. INTRODUCTION Molecular phylogenetic studies (Weiß & Oberwinkler 2001) have revealed that the heterobasidiomycetous family Sebacinaceae does not belong to the Auriculariales, a group of wood-decaying fungi in which it had been placed mainly on the basis of ultrastructural and microscopical characters (Bandoni 1984). This was a surprise since species of the Sebacinaceae are morphologically very similar to members of the Auriculariales with which they share, for example, the longitudinally septate basidia. Subsequently a growing number of DNA sequences derived from plant roots were * Part 221 of the series Studies in Heterobasidiomycetes from the Botanical Institute, University of Tübingen, Tübingen. ** Corresponding author. published that can be assigned to the Sebacinaceae; it became evident that members of this family are involved in a wide spectrum of mycorrhizal types : ectomycorrhizas (Glen et al. 2002, Selosse, Bauer & Moyersoen 2002, Tedersoo et al. 2003, Urban, Weiß & Bauer 2003), orchid mycorrhizas (McKendrick et al. 2002, Selosse et al. 2002, Taylor et al. 2003), ericoid mycorrhizas (Allen et al. 2003), and even in jungermannioid mycorrhizas, an only recently described association with liverworts (Kottke et al. 2003). Up to then in heterobasidiomycetes a mycorrhizal potential was only known from some taxa that occur as orchid symbionts (Rasmussen 2002). The broad diversity of mycorrhizal strategies present in Sebacinaceae is unique. This study presents the results of comprehensive molecular phylogenetic analyses using the nuclear gene for the ribosomal large subunit (nrLSU) that Sebacinales, overlooked mycorrhizal heterobasidiomycetes 1004 shed light on the ecology and evolution of a fascinating group of fungi whose striking biodiversity and ecological importance has only recently started to be recognised. Table 1. Strains of Sebacina vermifera included in this study. These strains were isolated from plant roots, grown in pure culture, and determined by induction of their basidial stages by J. H. Warcup (Warcup 1988). MATERIAL AND METHODS Warcup isolate no. GenBank accession no. (nrLSU) Host plant 140 714 723 750 768 914 915 963 977 AY505548 AY505549 AF291366 AY505550 AY505551 AY505552 AY505553 AY505554 AY505555 Eriochilus scaber (Orchidaceae) Eriochilus scaber (Orchidaceae) Cyrtostylis reniformis (Orchidaceae) Caladenia catenata (Orchidaceae) Glossodia minor (Orchidaceae) Phyllanthus calycinus (Euphorbiaceae) Caladenia catenata (Orchidaceae) Microtis uniflora (Orchidaceae) Microtis uniflora (Orchidaceae) Sample sources, DNA extraction, PCR and sequencing DNA sequences that were determined for this study were obtained from fungal herbarium specimens, from mycorrhizas of different types, or from axenic fungal cultures. Extraction of genomic DNA, PCR amplification and sequencing of the nuclear coded D1/D2 region of the ribosomal large subunit was performed as described elsewhere : Weiß & Oberwinkler (2001) for dried reference collections and axenic fungal cultures, Selosse et al. (2002) for the mycorrhizas of the terrestrial orchids Epipactis helleborine and E. microphylla, and Selosse et al. (2002) and Urban, Weiß & Bauer (2003) for ectomycorrhizas. Provenance and plant hosts of the mycorrhizal samples are indicated in Fig. 2. We were pleased to be able to include in this study some of the original Warcup Sebacina vermifera strains, mostly isolated from Australian orchids (Warcup 1988) ; details of these strains are provided in Table 1. Phylogenetic analysis These sequences were analysed together with sequences already available in GenBank (http://www.ncbi.nlm. nih.gov/) ; accession nos are given in Figs 1 and 2. We analysed two data sets : (1) 65 sequences covering the major groups of basidiomycetes to estimate the phylogenetic placement of the Sebacinaceae ; and (2) 107 sebacinoid sequences to elucidate phylogenetic relationships within the Sebacinaceae. Alignments were constructed using CLUSTAL X (Thompson et al. 1997) and manually edited with Se-Al (Rambaut 1996). Ambiguous alignment positions were excluded from the phylogenetic analyses. To estimate phylogenetic relationships, alignments were analysed using a Bayesian approach based on Markov chain Monte Carlo (MCMC) as implemented in MrBayes 3.0b4 (Huelsenbeck & Ronquist 2001). With this method it is possible to estimate a posteriori probabilities for the monophyly of given groups, i.e. the probability that a group is monophyletic given the DNA alignment. For each alignment we ran four incrementally heated simultaneous Monte Carlo Markov chains over ten million generations using the general time-reversible model of DNA substitution, additionally assuming a percentage of invariable alignment sites with gammadistributed substitution rates of the remaining sites (GTR+I+G; see Swofford et al. 1996), and random starting trees. Trees were sampled every 100 generations resulting in an overall sampling of 100 000 trees, from which the last 60 000 trees were used to compute a 50 % majority rule consensus tree to get estimates for the posterior probabilities. Stationarity of the chains was controlled using the Tracer software, version 1.0 (Rambaut & Drummond 2003). With the same software we calculated mean values for the parameters of the DNA substitution model that were sampled during the MCMC process (again using the last 60 000 samples). These mean values were then used to estimate branch lengths of the consensus trees with PAUP 4.0b10 (Swofford 2002) via maximum likelihood. To avoid possible pitfalls of the MCMC approach (Huelsenbeck et al. 2002), we repeated the MCMC analysis for each alignment four times, always starting with random trees. We also performed neighbourjoining analyses (NJ ; Saitou & Nei 1987) using Kimura 2-parameter distances (Kimura 1980), combined with non-parametric bootstrap analyses (Felsenstein 1985) in PAUP. RESULTS AND DISCUSSION The results of the different runs of MCMC on the same alignment yielded very similar results, only differing slightly in posterior probability values. Stationarity of the Markov chains was obviously reached before 10 000 trees (alignment 1) and 20 000 trees (alignment 2) had been sampled. Figs 1 and 2 each present the consensus of one of the four MCMC analyses performed on each alignment. The results of the bootstrapped NJ analyses (data not shown) were widely consistent to the results of the MCMC analyses, i.e. groups supported by bootstrap values exceeding 50 % were generally not in conflict with groupings obtained by MCMC. An exception is the placement of Urediniomycetes and Ustilaginomycetes that appeared as sister groups in the NJ analysis. Bootstrap support was generally lower in the NJ analyses than the corresponding posterior probabilities inferred from MCMC analyses. Basidiomycete phylogeny Compared to other published molecular phylogenetic hypotheses concerning higher-level relationships in Cortinarius mussivus AF291307 75 72 Tricholoma vaccinum AF291378 96 Kuehneromyces mutabilis AF291342 Agaricus augustus AF291286 0.05 substitutions/site 100 100 Amanita muscaria AF024465 Boletus edulis AF291300 85 100 Coniophora olivacea AF098376 73 Amphinema byssoides AF291288 Russula cyanoxantha AF291361 68 98 Echinodontium tinctorium AF393056 Homobasidiomycetes 93 Fomes fomentarius AF291331 Polyporus varius AF291356 Inonotus nodulosus AF291341 81 100 Hymenochaete rubiginosa AF291339 Basidioradulum radula AF291299 Thelephora palmata AF291265 Ramaria stricta AF287887 65 100 Phallus impudicus AY152404 Botryobasidium subcoronatum AF287850 Multiclavula mucida AF287875 100 87 Tulasnella calospora AY152407 Ceratosebacina calospora AF291304 Tulasnella/Ceratobasidium Ceratobasidium cornigerum AY152405 Dacrymyces stillatus AF291309 99 100 99 Calocera viscosa AF011569 Dacrymycetales Femsjonia peziziformis AF291330 58 Exidia glandulosa AF291319 69 Exidiopsis calcea AF291326 Auricularia auricula-judae AF291289 59 Bourdotia galzinii AF291301 100 99 Basidiodendron eyrei AF291296 Auriculariales 84 Endoperplexa enodulosa AY505543 Stypella vermiformis AF291369 96 86 Hyaloria pilacre AF291338 100 Myxarium nucleatum AF291351 Sebacina incrustans AF291365 78 94 Tremelloscypha gelatinosa AF291376 100 Sebacina dimitica AF291364 98 Efibulobasidium rolleyi AF291317 Sebacinales 99 Craterocolla cerasi AF291308 100 Sebacina allantoidea AF291367 100 Sebacina vermifera AF291366 76 Geastrum saccatum AF287859 Tremella mesenterica AF011570 79 Tremella foliacea AF291373 88 Sirobasidium magnum AF042241 Filobasidiella neoformans AF075484 99 Tremellomycetidae Cuniculitrema polymorpha AY032662 99 99 Trichosporon cutaneum AF075483 100 Holtermannia corniformis AF189843 Filobasidium floriforme AF075498 Thecaphora seminis-convolvuli AF009874 100 100 Urocystis ranunculi AF009879 Ustilago hordei L20286 66 Tilletia caries L20285 Ustilaginomycetes 100 Georgefischeria riveae AF009861 Exobasidium vaccinii AF009858 87 Doassansia epilobii AF007523 Eocronartium muscicola L20280 100 Septobasidium carestianum L20289 Urediniomycetes II Puccinia graminis L08721 50 Aurantiosporium subnitens AF009846 100 Ustilentyloma fluitans AF009882 Urediniomycetes I Microbotryum violaceum AF009866 Taphrina deformans U94948 Hymenomycetes 1005 Hymenomycetidae M. Weiss and others Fig. 1. Phylogenetic placement of Sebacinales within the basidiomycetes : Bayesian Markov chain Monte Carlo analysis of an alignment of nuclear DNA sequences from the D1/D2 region of the large ribosomal subunit. The topology was rooted with the ascomycete Taphrina deformans. Numbers on branches are estimates for a posteriori probabilities that the respective groups are monophyletic given the data. For more details see the text. basidiomycetes (e.g. Swann & Taylor 1993, 1995, Gargas et al. 1995, Begerow, Bauer & Oberwinkler 1997) the present MCMC analysis of alignment 1 (Fig. 1) offers a high resolution, especially in the backbone of the phylogenetic tree. Thus, with a posterior probability of 99 %, rust fungi and their relatives (Urediniomycetes I and II in Fig. 1) are basal in the basidiomycetes. Smut fungi (Ustilaginomycetes) and Hymenomycetes together form a monophyletic group, possibly with the type B secondary structure of the 5S rRNA as an apomorphy (Gottschalk & Blanz 1985). Sebacinales, overlooked mycorrhizal heterobasidiomycetes 83 OM Neottia nidus-avis AF465190 F OM Neottia nidus-avis + ECM AF440657 F 89 OM Neottia nidus-avis + ECM AF440641 F OM Neottia nidus-avis + ECM AF440643 F OM Neottia nidus-avis + ECM AF440644 F Sebacina aff. epigaea AF291363 D 59 ECM Dryas octopetala AY452680 N OM Neottia nidus-avis + ECM AF440649 F OM Neottia nidus-avis AF440662 F OM Neottia nidus-avis AF440663 F 75 OM Neottia nidus-avis + ECM AF440654 F Tremellodendron sp. AY505547 USA Orchid mycorrhiza (OM) 100 72 OM Neottia nidus-avis AF440655 F Ectomycorrhiza (ECM) OM Hexalectris spicata AY243515 USA ECM Salix sp. AY452682 N Ericoid mycorrhiza (ERM) OM Neottia nidus-avis + ECM AF440652 F Jungermannioid mycorrhiza (JM) 75 OM Epipactis helleborine AY452678 F OM Epipactis helleborine AY452677 F ECM Populus tremula AJ534931 EST 82 94 OM Neottia nidus-avis AF440651 F OM Neottia nidus-avis + ECM AF440656 F 80 OM Hexalectris spicata AY243518 USA OM Neottia nidus-avis AY052372 D 52 ECM Dryas octopetala AY452681 N OM Neottia nidus-avis + ECM AF440650 F OM Neottia nidus-avis AF440660 F A Austria 99 OM Neottia nidus-avis + ECM AF440658 F AUS Australia OM Neottia nidus-avis + ECM AF440646 F OM Neottia nidus-avis + ECM AF440659 F CAN Canada OM Epipactis microphylla AY286192 F CHN P.R. China Sebacina sp. AY505544 USA OM Neottia-nidus avis + ECM AF440648 F D Germany OM Neottia-nidus avis + ECM AF440647 F 67 E Spain OM Hexalectris revoluta AY243517 USA ECM Picea abies AJ534930 EST EST Estonia Sebacina dimitica AF291364 D 72 55 F France ECM Pinus sylvestris AY505562 A 98 Tremellodendron sp. AY505546 USA IND India Tremellodendron pallidum AF384862 CAN OM Hexalectris spicata AY243516 USA MEX Mexico 99 ECM Eucalyptus marginata AY072814 AUS N Norway (Svalbard) 69 OM Neottia nidus-avis + ECM AF440653 F 60 OM Epipactis helleborine AY452674 F OM Neottia nidus-avis AY052373 UK 100 Sebacina cf. epigaea AY505560 A OM Epipactis helleborine AY452676 F 100 98 100 OM Epipactis helleborine AY452679 F ECM Tilia sp. AF509966 A 100 Sebacina incrustans AY143340 D 100 ECM Picea abies AF509967 A Sebacina incrustans AF291365 D 71 Sebacina sp. AF465191 F 81 100 0.005 substitutions/site 83 Sebacina sp. AY505558 A Sebacina cf. incrustans AY505561 A 100 Sebacina sp. AF465185 F Sebacina sp. AF440664 F 100 Sebacina incrustans AY505545 CHN 100 ECM Corylus colurna AY505563 A 100 Sebacina epigaea AF291267 D 87 Sebacina cf. epigaea AY505559 A ECM Tilia cordata AJ534932 EST Tremelloscypha gelatinosa AF291376 MEX 63 100 OM Epipactis helleborine AY452675 F 87 ECM Eucalyptus marginata AY093436 AUS ECM Eucalyptus marginata AY093438 AUS 100 Efibulobasidium rolleyi AF291317 CAN 100 Craterocolla cerasi AY505542 D 100 100 Craterocolla cerasi AF291308 D Efibulobasidium albescens AF384860 CAN Sebacina allantoidea AF291367 D ERM Gaultheria shallon AF300777 CAN ERM Gaultheria shallon AF300784 CAN 95 ERM Gaultheria shallon AF300774 CAN ERM Gaultheria shallon AF300775 CAN ERM Gaultheria shallon AF300783 CAN ERM Gaultheria shallon AF300782 CAN 100 ERM Gaultheria shallon AF300781 CAN ERM Gaultheria shallon AF300778 CAN ERM Gaultheria shallon AF300780 CAN ERM Gaultheria shallon AF300779 CAN 94 ERM Gaultheria shallon AY112930 CAN ERM Gaultheria shallon AF300776 CAN 100 ERM Gaultheria shallon AF300785 CAN ERM Gaultheria shallon AF284137 CAN 100 99 ERM Gaultheria shallon AF300786 CAN ERM Gaultheria shallon AF300787 CAN ERM Gaultheria shallon AF300790 CAN 98 ERM Gaultheria shallon AF284136 CAN 99 ERM Gaultheria shallon AF300788 CAN ERM Gaultheria shallon AF300789 CAN 98 67 ERM Gaultheria shallon AF300791 CAN 87 100 ERM Gaultheria shallon AF300793 CAN ERM Gaultheria shallon AF300792 CAN 59 JM Lophozia sudetica AY298946 E 96 91 JM Lophozia incisa AY298847 E JM Calypogeia muelleriana AY298948 F Sebacina vermifera (OM) AF291366 AUS 96 Sebacina vermifera (OM) AY505549 AUS 85 100 Sebacina vermifera AY505552 AUS Sebacina vermifera (OM) AY505555 AUS Multinucleate rhizoctonia AY505556 AUS 100 69 82 Piriformospora indica AY505557 IND Sebacina vermifera (OM) AY505554 AUS 100 97 Sebacina vermifera (OM) AY505548 AUS 95 Sebacina vermifera (OM) AY505551 AUS Sebacina vermifera (OM) AY505550 AUS Sebacina vermifera (OM) AY505553 AUS Geastrum saccatum AF287859 Fig. 2. For legend see opposite page. 1006 A B M. Weiss and others Our study confirms that it is difficult to separate homobasidiomycetes, as currently defined, from other hymenomycetous taxa such as Tulasnellales and Ceratobasidiales (Hibbett & Thorn 2001, Weiß, Bauer & Begerow 2004). In our analysis, Tulasnella calospora groups as highly supported with the lichen-forming homobasidiomycete Multiclavula mucida, representing the cantharelloid clade sensu Hibbett & Thorn (2001). This position is consistent with other molecular phylogenetic analyses (e.g. Bruns et al. 1998, Hibbett, Gilbert & Donoghue 2000, Bidartondo et al. 2003). Also with respect to other homobasidiomycetous clades, our results are consistent with clades recognised in Hibbett & Thorn (2001), with one exception: Geastrum was separated from the gomphoid-phalloid clade, which in our analysis is represented by species of Ramaria and Phallus, and appears as a sister taxon of the Sebacinaceae (see the discussion below). Phylogenetic position of the Sebacinaceae With a high posterior probability, the Sebacinaceae occupy a basal position within the Hymenomycetidae, with Geastrum as a sister group (Fig. 1). A close phylogenetic relationship between the Sebacinaceae and Geastrum has recently also been found in another molecular phylogenetic analysis of nrLSU D1/D2 sequences (Taylor et al. 2003). Considering that Geastrum is also capable of forming mycorrhizas (Agerer & Beenken 1998), and the more basal groups in basidiomycetes mainly include mycoparasitic and plant parasitic fungi (Weiß, Bauer & Begerow 2004), we hypothesise that the common ancestor of this Geastrum/ Sebacinaceae clade, or even the common ancestor of the whole group of the Hymenomycetidae, was ectomycorrhizal. If this assumption of an apomorphic mycorrhizal status in Hymenomycetidae holds, then the distribution of mycorrhizal taxa within the homobasidiomycetes could be explained by multiple independent origins of saprotrophism rather than by convergent evolution of mycorrhizas, in contrast to current hypotheses on the evolution of ectomycorrhizas in basidiomycetes (e.g. Hibbett, Gilbert & Donoghue 2000). Regarding the phylogenetic position of the Sebacinaceae within the basidiomycetes (Fig. 1), which is corroborated by the ecological data at hand, it is appropriate to establish a new basidiomycetous order : Sebacinales M. Weiß, Selosse, Rexer, A. Urb. & Oberw., ordo nov. Fungi Hymenomycetum. Basidia longitudinaliter septata. Septa doliporis parenthesomatibus imperforatis, efibulata. Cystidia nulla. 1007 Typus ordinis: Sebacinaceae Oberw. & K. Wells 1982 (in Wells & Oberwinkler 1982: 329). This new order can be morphologically separated from species of Auriculariales, in which the Sebacinaceae has been placed up to now (Bandoni 1984), by a combination of longitudinally septate basidia, imperforate parenthesomes at the septal pores, and a lack of both clamp connections and cystidia. The lack of cystidia has to be included in this diagnosis since Endoperplexa enodulosa, a species which according to our molecular phylogenetic analyses does not belong to the Sebacinales (Fig. 1), differs from our description of Sebacinales only in the presence of cystidia (Roberts 1993). Consequently, the order Auriculariales has to be emended to include saprotrophic hymenomycetes with septate basidia and septa with imperforate parenthesomes, where cystidia are present in species that lack clamp connections. Unfortunately, this emendation is not perfectly congruent with our molecular phylogenetic analysis. It is, in the literal meaning of the word, an improvement in the exclusion of Sebacinales, but not the ultimate solution, as there are taxa such as Ceratosebacina calospora or Exidiopsis gloeophora that fit the emended concept of Auriculariales, but obviously should be excluded from that group according to molecular phylogenetic results (Fig. 1; Weiß & Oberwinkler 2001). At the moment, we see no way to solve this problem. Hopefully other characters will be detected in the future that will allow a more elegant morphological circumscription of both Auriculariales and Sebacinales. Phylogenetic relationships within Sebacinales The MCMC hypothesis of phylogenetic relationships in the Sebacinales shows a division into two subgroups referred to here A and B (Fig. 2). Group A contains all the sequences obtained from basidiomes, from ectomycorrhizas, and sebacinoid mycobionts of the orchids Neottia nidus-avis, Epipactis and Hexalectris (i.e. of at least partly heterotrophic orchids ; Taylor et al. 2003, Selosse et al. 2004). Sebacina and Tremellodendron appear to be polyphyletic. Subgroup B contains in basal positions various sequences of Sebacina vermifera that were obtained from axenic fungal cultures, mostly originating from the roots of green, autotrophic Australian orchids (Warcup 1988; Table 1). The three sebacinoid sequences from liverwort rhizoids included in this study (Kottke et al. 2003) appear as a monophyletic group, which according to our MCMC analysis (Fig. 2) represents the sister group to the one containing the sebacinoid rDNA sequences from ericoid mycorrhizas Fig. 2. Phylogenetic relationships within Sebacinales : Bayesian Markov chain Monte Carlo analysis of an alignment of nuclear DNA sequences from the D1/D2 region of the large ribosomal subunit. The topology was rooted with Geastrum saccatum. Numbers on branches are estimates for a posteriori probabilities that the respective groups are monophyletic given the data. The two main subgroups of Sebacinales discussed in the text are designated A and B. Sequences from teleomorphic specimens are printed in bold. Sebacinales, overlooked mycorrhizal heterobasidiomycetes of Gaultheria shallon (Allen et al. 2003). All teleomorphs known for species in group B were observed only in axenic culture and morphologically assigned to Sebacina vermifera (Warcup 1988). 1008 herbarium material of S. incrustans (Lowy 1971 ; Peter Roberts, pers. comm.), we suggest that the Sebacinales has a wide distribution. Mycorrhizal diversity Generic and species concepts in Sebacinales Our results indicate that basidiome gross morphology is not useful for the definition of monophyletic groups in Sebacinales, a statement which obviously can be generalised for wide parts of a natural systematic concept in basidiomycetes (e.g. Oberwinkler 1977, Bandoni 1984, Hibbett & Thorn 2001). Thus, Sebacina, defined for resupinate forms (Tulasne & Tulasne 1871), is polyphyletic according to the present analysis. A morphological transition from certain species of Sebacina to Tremellodendron, a genus name introduced for clavarioid species, has been described (McGuire 1941). Our analysis supports this point of view. Efibulobasidium, a genus based on pustulate basidiomes (Wells 1975), is, according to our results, another example of a probably polyphyletic group circumscribed by basidiome morphology. Not only the generic concept, but also the species concepts in Sebacina appear to be questionable. The molecular analysis shows that diverse species might be included in the present circumscription of Sebacina incrustans, the type of Sebacina ; the same situation holds for S. epigaea. The problem for an accurate delimitation of species is the lack of useful macroand microscopical characters. Obviously, biodiversity in this group is much higher than hitherto assumed. This is corroborated by the molecular diversity detected in mycobionts of ectomycorrhizas or orchid mycorrhizas, from which up to now no data about sexual stages are available. Judging from our molecular phylogenetic hypotheses, most of the mycorrhizal mycobionts contained in subgroup A should morphologically be classified in Sebacina or Tremellodendron. There is a particularly high probability that one of the Neottia mycobionts (AF440655), sequenced from roots of a French specimen of N. nidus-avis (Selosse et al. 2002), is a Tremellodendron species, since its D1/D2 sequence is identical with that obtained from a Tremellodendron sample from North America. If they are conspecific, this would be the first molecular evidence for a wide geographical distribution of a single species of the Sebacinales. It is, however, premature to speculate about geographical distribution patterns of sebacinoid species, since the present sampling of DNA sequences of Sebacinales is strongly biased on collections from Europe, Australia (Sebacina vermifera and mycobionts of Eucalyptus) and North America (Gaultheria mycobionts). From the limited molecular data, however, we can infer that both Sebacinales subgroups A and B are distributed over Europe, Australia, and North America. In our analysis we were able to also include a Chinese specimen of the S. incrustans complex. Judging from In the field, ectomycorrhizas involving Sebacinales mycobionts have only been well documented for group A (Glen et al. 2002, Selosse, Bauer & Moyersoen 2002, Urban, Weiß & Bauer 2003). The potential to form ectomycorrhizas in vitro has been demonstrated for species of the Sebacina vermifera complex (Warcup 1988), but it is not clear whether such associations also occur in the field. On the other hand, orchid mycorrhizal species of Sebacinales occur in both subgroups A and B (Warcup 1988, McKendrick et al. 2002, Selosse et al. 2002, Taylor et al. 2003), but they may not be homologous. In subgroup A, sebacinoid mycobionts colonise achlorophyllous species, such as Neottia nidusavis and Hexalectris spicata (Selosse et al. 2002, Taylor et al. 2003), or green Epipactis species for which achlorophyllous individuals exist and that are likely to be partially heterotrophic (Selosse et al. 2004). These orchid mycobionts also form ectomycorrhizas with diverse surrounding trees (Selosse, Bauer & Moyersoen 2002, Selosse et al. 2002, 2004), suggesting a tripartite association, where the orchid derives resources from the tree via the sebacinoid mycobiont, as described for other achlorophyllous orchids (e.g. Taylor & Bruns 1997, McKendrick, Leake & Read 2000). It is therefore possible that all of the sebacinoid orchid mycobionts of subgroup A have ectomycorrhizal potential. Species of the Sebacina incrustans complex cannot be grown in pure culture (F. O., unpubl.), which may be indicative of a strictly ectomycorrhizal life strategy for species belonging to group A. On the other hand, orchid symbionts of group B have been isolated and grown in pure culture (Warcup & Talbot 1967, Warcup 1988). Only these species, and not the orchid symbionts found in group A, may belong to the highly polyphyletic form genus Rhizoctonia (Milligan & Williams 1987, Taylor et al. 2003). The Sebacina vermifera complex The present molecular phylogenetic study includes fungal isolates that have been assigned to Sebacina vermifera (Warcup & Talbot 1967, Warcup 1988). These isolates were obtained from roots of Australian orchids and it was shown that some isolates were able to stimulate the germination of orchid seeds and to form ectomycorrhizas with myrtaceous species in vitro (Warcup 1988). Concerning his isolates of S. vermifera, which varied in microscopical measurements as well as in growth parameters of axenic cultures, Warcup (1988) states that ‘without further data it is difficult to decide whether S. vermifera is a variable species or a complex of closely allied species. ’ Our analyses strongly suggest that S. vermifera is indeed a broad complex of species M. Weiss and others (Fig. 2). It is even possible that all the species that can currently be assigned to Sebacinales subgroup B belong to this morphologically defined complex, since the liverwort mycobionts (Kottke et al. 2003) and those of Gaultheria shallon (Berch, Allen & Berbee 2002, Allen et al. 2003), which are also included in subgroup B, have up to now only been detected by molecular or ultrastructural means, and nothing is known about their sexual stages. We also included in the present study two isolates obtained from arbuscular mycorrhizas (AM); the multinucleate rhizoctonia DAR 29830 was isolated from a vesicle of Glomus fasciculatum (Williams 1985, Milligan & Williams 1987) and the anamorphic Piriformospora indica isolated from a spore of Glomus mosseae (Verma et al. 1998). Both isolates are closely related according to our molecular phylogenetic analysis, which is consistent with their morphological characters (Milligan & Williams 1987, Varma et al. 2001), and positioned among isolates of the Sebacina vermifera complex (Fig. 2). Similar isolates were frequently obtained in Australia from pot cultures of AM, but also from diverse host plants in the field (Milligan & Williams 1987). So far nothing is known about the specific diversity of these organisms, nor do we have data about their interaction with AM fungi, but it was shown that the isolate designated as Piriformospora indica was able to benefit plant growth and increase resistance against pathogens in a broad range of host plants (Varma et al. 2001). This phylogenetic analysis shows a broad mycorrhizal capacity in Sebacinales, including ectomycorrhizas (Warcup 1988, Glen et al. 2002, Selosse, Bauer & Moyersoen 2002, Selosse et al. 2002), orchid mycorrhizas (Warcup 1988, McKendrick et al. 2002, Selosse et al. 2002), ericoid mycorrhizas (Allen et al. 2003), and also the recently recognised jungermannioid mycorrhizas (Kottke et al. 2003). Despite the tremendous increase of data in recent years, sampling of Sebacinales, both geographically and with respect to the host plants, is still erratic. The present data are but the tip of an iceberg, as corroborated by a recent quantitative study (Avis et al. 2003), in which 5% of the ectomycorrhizas in a temperate oak savanna were ascribed to Sebacinales. Future studies will bring more detailed insight into the ecology and phylogeny of this fascinating fungal order, which may have a future economically important plant-beneficial potential, which has hitherto been overlooked. ACKNOWLEDGEMENTS We thank Roland Kirschner, Volker Kummer, Peter Roberts, Ajit Varma and Philip Williams and the National Institute of Agrobiological Sciences (Japan) for the loan of specimens or cultures, Anders Dahlberg, Monique Gardes and Morag Glen for sharing unpublished DNA sequences, Duur Aanen, Robert Bauer, Martin Bidartondo, Ruth Fleischmann and Sigisfredo Garnica for critically reading earlier drafts of the manuscript and helpful discussions, and 1009 Jacqueline Götze and Annie Tillier for technical assistance. 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