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University Innsbruck, Institute of Microbiology, Technikerstr. 25, 6020 Innsbruck, Austria
Jean-Marc Moncalvo
Centre for Biodiversity and Conservation Biology, Royal Ontario Museum, 100 Queen’s Park, Toronto, Ontario, M5S 2C6
Rytas Vilgalys
Department of Biology, Duke University, Durham, North Carolina 27708
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Research on the molecular systematics of Cortinarius, a species-rich mushroom genus with nearly global distribution, is just beginning. The present study explores infrageneric relationships using rDNA ITS and LSU sequence data. One large dataset of 132 rDNA ITS sequences and one combined da-taset with 54 rDNA ITS and LSU sequences were generated. Hebeloma was used as outgroup. Bayesian analyses and maximum-likelihood (ML) analyses were carried out. Bayesian phylogenetic inference performed equally well or better than ML, especially in large datasets. The phylogenetic analysis of the combined dataset with species representing all currently recognized subgenera recovered seven well-supported clades (Bayesian posterior probabilities BPP > 90%). These major clades are: /Myxacium s.l., /subg. Cortinarius, the /phlegmacioid clade (including the subclades /Phlegmacium and /Delibuti), the /calochroid clade (/Calochroi, /Ochroleuci and /Allutus), the /telamonioid clade (/Telamonia, /Orellani, /Anomali), /Dermocybe s.l. and /Myxotelamonia. Our results show that Cortinarius consists of many lineages, but the relationships among these clades could not be elucidated. On one hand, the low divergence in rDNA sequences can be held responsible for this; on the other hand, taxon sampling is problematic in Cortinarius phylogeny. Because of the incredibly high diversity (~2000 Cortinarius species), our sampling included <5% of the known species. By choosing type species of subgenera and sections, our sampling is strongly biased toward Northern Hemisphere taxa. More extensive taxon sampling, especially of species from the Southern Hemisphere, is essential to resolve the phylogeny of this important genus of ectomycorrhizal fungi.
Key words: Bayesian analysis, ITS phylogeny, maximum-likelihood analysis, taxon sampling
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). These characters delimit Cortinarius taxa: (i) a rusty brown spore print and warty-rough spore ornamentation, (ii) spores lacking a germ pore or loosening perisporium, (iii) and a cortinate inner veil. Cortinarius spp. form mycorrhizal associations with ectotrophic trees (Singer 1986). Macroscopically, members of this genus are highly variable: their habit varies from mycenoid to tricholomatoid; pilei can be dry, squamose, silky, fibrillose or viscid; the outer veil can vary from very fugacious to persistently belt-like or entirely mucilaginous; and fruit bodies can be colorful or uniformly dull brown.
The subdivision of Cortinarius into subgeneric units causes problems because of the overall considerable morphological variation in this genus: different manifestations of each character have been observed between, and sometimes even within, species. This difficulty in defining character states, as well as the different weights assigned to such morphological characters by different taxonomists, has been the largest obstacle to the establishment of a broadly acceptable Cortinarius classification. The many controversies (Kühner and Romagnesi 1953, Orton 1958, Kühner 1980, Moser 1983, Moser in Singer 1986, Melot 1990, Moënne-Loccoz et al 1990) have resulted in a taxonomic chaos and indicate that morphology alone is insufficient for recognizing natural units in this group of fungi.
After the discovery of striking pigments and secondary compounds in Dermocybe and Leprocybe (Kopanski et al 1982, Steglich and Oertel 1984, Arnold et al 1987, Gill and Steglich 1987, Arnold 1993, Gill 1995a, b), Cortinarius taxonomists had great expectations for chemotaxonomy. These hopes have been fulfilled only partly. Molecular phylogenetics is now regarded as more promising for studying the evolution in this complex genus. Earlier molecular investigations of Cortinarius, based mainly on sequences of the rDNA internal transcribed spacer (ITS) or the rDNA large subunit (LSU), have implied that Cortinarius is monophyletic (Liu et al 1997, Høiland and Holst-Jensen 2000, Moncalvo et al 2000, Peintner et al 2001). But the monophyly of all currently defined larger subgenera (>10 species), namely Dermocybe, Leprocybe, Myxacium, Phlegmacium, Sericeocybe and Telamonia has been rejected (Liu et al 1997, Chambers et al 1999, Høiland and Holst-Jensen 2000, Seidl 2000, Peintner et al 2002c, Garnica et al 2003b). Our own earlier phylogenetic studies demonstrated that the Cortinarius clade also included the three morphologically similar genera, Rozites, Cuphocybe and Rapacea, as well as the sequestrate genera Thaxterogaster, Protoglossum and Quadrispora (Peintner et al 2001, 2002).
The aim of the present study is to gain a better understanding of the phylogenetic relationships in Cortinarius. The effect of taxon sampling versus character sampling is examined and discussed briefly. Our reconstruction of phylogenetic relationships within Cortinarius is based on a large (132 taxa) ITS dataset. An additional rDNA dataset with 54 combined ITS and LSU sequences of representative taxa also was used for this purpose. Both datasets were analyzed using two analytical approaches: maximum likelihood (ML) and Bayesian inference of phylogeny.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) were sampled, including a broad selection of diverse Cortinarius taxa (including Dermocybe, Rozites, Cuphocybe and Thaxterogaster) from the Southern Hemisphere. Type specimens or specimens collected near the type locality were used whenever possible. Species interpretations follow Moser (1960), Moser and Horak (1975) and Moser (1983). Two sampling strategies were followed: (i) 132 ITS sequences were used to produce a large, taxon-rich phylogeny, with representative species selected based on traditional taxonomy; and (ii) LSU sequences were sampled for representative species from each terminal clade recovered by the ITS phylogeny.
The genus Hebeloma was chosen as outgroup to root the Cortinarius phylogenies, because earlier studies have shown Hebeloma to be a possible sister group (Peintner et al 2001, Moncalvo et al 2002). Sampled taxa, voucher specimen, geographical locations and Genbank accession numbers are listed in TABLE I. Species names follow the nomenclature of Garnier (1991). Recently recognized Cortinarius names are used for former Thaxterogaster, Rozites, Cuphocybe and Rapacea epithets (Peintner et al 2002a, b, Kuhnert and Peintner 2003). To distinguish between clade names and formal taxonomic epithets, clade names are not italicized and preceded with the symbol "/" (Moncalvo et al 2002).
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were followed. Primers used for PCR amplification and sequencing of the ITS region were ITS1, ITS1F, ITS2, ITS3 and ITS4 (White et al 1990, Gardes and Bruns 1993). The 5' end of the nLSU gene was amplified with either LRØR –LR5 or LRØR –LR7 primer pairs. Sequencing primers were LRØR, LR3, LR3R, LR5 and LR16 (Vilgalys and Sun 1994, Hopple and Vilgalys 1999, Moncalvo et al 2000). Amplification protocols followed conditions described by Vilgalys and Hester (1990).
Phylogenetic analyses. –
Sequences were aligned manually and regions of ambiguous alignment were excluded from the analyses. Gaps were treated as missing data. The program Modeltest version 3.06 (Posada and Crandall 1998) was used to choose the best-fit model of DNA substitution for each dataset, as determined from the Akaike information criterion (AIC). Maximum-likelihood (ML) analyses were carried out with the computer program PAUP* 4.0b10 (Swofford 2001) and conducted with "asis" addition sequence and TBR branch swapping. Branch support was assessed by bootstrapping with simple taxon addition, NNI, with 100 replicates, five trees held at each step (Salamin et al 2003).
The Bayesian approach to phylogenetics allows simultaneous estimation of the uncertainty associated with any parameter within a phylogenetic model (e.g., topology) by using posterior probabilities. For these complex estimation problems, Markov Chain Monte Carlo (MCMC; Largent and Simon 1999) methods were used to estimate the posterior distribution of parameters. Bayesian analysis was carried out with the computer program MrBayes 2.01 (Huelsenbeck and Ronquist 2001). Random trees were used as the starting point. Sample frequency and print frequency occurred once every 100 generations. To improve mixing of the chain, four simultaneous MCMC chains were run. Branch lengths of the trees were saved. For those trees that were sampled after the process had reached stationarity, a 50% majority-rule consensus tree was computed with PAUP* to get estimates for posterior probabilities. Bayesian consensus phylograms were calculated with MrBayes. Bayesian analysis was carried out three times to test the independence of the results from topological priors. Clades with Bayesian Posterior Probabilities (BPP) higher than 70% were named, as far as possible, in accordance with the nomenclature of Moser (Singer 1986). Because this represents a preliminary phylogenetic classification of Cortinarius, no formal epithets are applied to clade names.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. A starting tree for branch swapping was constructed by neighbor joining. The heuristic search was carried out with TBR branch swapping. The ML search was stopped after 30 d of computing time. Seven ML trees were obtained with a –Ln likelihood of 9811.8250.
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was calculated using 28 302 trees from the third Bayesian analysis. This longest chain reached stationarity at about 170 000 generations: the first 1700 trees were discarded as "burn in", and 28 302 were used to calculate the consensus tree using MrBayes. The consensus phylogram is based on mean branch lengths and posterior probabilities. Branch lengths are maximum-likelihood estimates and are scaled in terms of expected numbers of nucleotide substitutions per site. The posterior distribution of parameters is listed in TABLE II
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. One ML tree was obtained with a –Ln likelihood = 9296.19703.
Bayesian inference of combined ITS and LSU data was estimated as described above. MrBayes was run for 700 000, 2 000 000 and 3 000 000 generations. The third analysis reached stationarity at about 150 000 generations: the first 1500 trees were discarded as "burn in", and 28 502 were used for the consensus phylogram (FIG. 2). For the calculation of BPP, 46 504 trees were used. The posterior distribution of parameters is shown in TABLE II.
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Bayesian analysis of the ITS rDNA region (FIG. 1) showed Cortinarius to be monophyletic, supported by BPP of 100%. Fourteen subclades with three or more species were supported by BPP 
70%. Four species rich lineages (10 spp.: /Myxacium sensu lato (s.l.), /Phlegmacium, /Telamonia, /Dermocybe s.l.), two medium size (/Calochroi 9 spp., /Delibuti 6 spp.) and eight small lineages were recovered. The clades /Myxacium s.l., /Calochroi, /Telamonia, and four small lineages received BPP of 99% or 100%. Supported clades with >3 species of FIG. 1 are described top down:
/Myxacium s.l. (BPP 100, Bootstrap BS 57) consists of three subclades: /Myxacium s.str. (= subg. Myxacium section [sec.] Myxacium), /Defibulati (= subg. Myxacium sec. Defibulati, and /Cycneus (= C. cycneus and "C. magellanicus" described by Horak and Wood 1990). /Myxacium s.l. includes agaricoid and sequestrate Cortinarii (Quadrispora and former Thaxterogaster spp.).
/Calochroi (BPP 100, BS 76) includes species in Phlegmacium sections Fulvi (C. elegantior, C. flavaurora, C. fulvocitrinus), Calochroi (C. calochrous, C. arcuatorum), and Scauri (C. atrovirens, C. odorifer, C. sulphurinus).
/subg. Cortinarius (BPP 100, BS 100) includes the type species of the genus (C. violaceus) and other representatives of subg. Cortinarius.
/Leprocybe (BPP 99, BS 91) includes the type species of subg. Leprocybe (C. cotoneus), and morphologically closely related species (C. venetus, C. flavifolius).
/Phlegmacium (BPP 70, BS < 50) is a lineage of 14 species in different sections of subg. Phlegmacium: sec. Phlegmacium (C. multiformis, C. fraudulosus and C. saginus, the typus of subg. Phlegmacium), sec. Calochroi (C. glaucopus), sec. Coerulescentes (C. variicolor, C. coerulescens, C. subfoetidus, C. citriolens), and sec. Scauri (C. percomis, C. russeoides). Two secotioid taxa (Hymenogaster remyi, H. sublilacinus) also belong to this lineage.
/Purpurascentes (BPP 93, BS < 50) circumscribes taxa in subg. Phlegmacium sec. Scauri. This lineage was recovered by tree topologies in all analytical approaches but with little and varying support. A recent study including seven temperate species in the C. scaurus species complex demonstrated the monophyly of /Purpurascentes (Moser and Peintner 2002b).
/Allutus (BPP 100, BS < 50) is a lineage of species in Phlegmacium sec. Phlegmacium.
/Ochroleuci (BPP 86, BS 91) circumscribes species in subg. Myxacium sec. Ochroleuci.
/Delibuti (BPP 79, BS < 50) is a lineage of species, which have been classified in subg. Myxacium sections Delibuti, Cystidiferae and Archeri. When excluding C. rotundisporus, an Australian species of Myxacium, this clade is strongly supported (BPP 96, BS 51).
/Orellani (BPP 100, BS 99) includes species of subg. Leprocybe sec. Orellani.
/Telamonia (BPP 99, BS < 50) represents Cortinarius spp. in subg. Telamonia, and taxa in the subgenera Sericeocybe (C. alboviolaceus, C. traganus) and Leprocybe (C. gentilis, C. raphanoides, C. humicola), as well as in the former genus Rozites (C. purpurellus, C. ochraceoazureus).
/Dermocybe s.l. (BPP 72, BS < 50) circumscribes Dermocybe spp. from the northern and southern hemisphere. Subgenus Dermocybe is paraphyletic, as /Dermocybe s.l. also includes taxa in subgenera Cystogenes (C. teraturgus) and Telamonia (C. firmus). Northern hemisphere taxa of Dermocybe form the well-supported (BPP 90, BS 86) subclade /Dermocybe s.str. (C. croceus, C. semisanguineus, C. sanguineus, D. marylandensis). Tree topologies indicate a sister group relationship of /Dermocybe s.l. with two clades of the Southern Hemisphere Dermocybe spp., /Icterinula and /Austronanceiensis.
/Acutus (BPP 87, BS 51) represents species in subg. Telamonia sec.Obtusi. The mycenoid basidiome habit, a inflated hyphae in the mediostratum of the lamellar trama, and the presence of cystidia distinguish species belonging to the Obtusi from other Telamonia spp.
/Myxotelamonia (BPP 87, BS < 50) consists of species in subg. Phlegmacium, sec. Amarescentes (C. infractus) and in subg. Telamonia sec. Myxotelamonia (C. cinereobrunneus). A gelatinized epicutis and grayish tints to the lamellae characterize both.
When comparing tree topologies obtained by the ML and the Bayesian analysis of the ITS dataset, the same larger clades are recovered. In general, BPP are higher than bootstrap (BS) values (FIG. 1). As clades become larger, BS values quickly decrease to below 50%, while BPP higher than 70% are still obtained (e.g., /Calochroi /Dermocybe s.l., /Myxacium s.l., /Phlegmacium, /Telamonia).
In the Bayesian estimation of the combined ribosomal DNA phylogeny (ITS and LSU), 10 clades were supported by BPP of at least 70% (FIG. 2). Also in this dataset, BPP generally were higher than BS values. Compared to the ML tree, three new relationships were supported (BPP > 85%): (i) the /phlegmacioid group, a lineage of /Delibuti and C. multifomis representing /Phlegmacium from the ITS phylogeny; (ii) the common lineage of /Calochroi, /Ochroleuci and /Allutus, tentatively addressed as the /calochroid group; (iii) the /telamonioid group, a common lineage of /Telamonia, C. anomalus, C. laetus and C. orellanus. Moreover, little support (BS 70, BPP < 50) is obtained for a larger /Purpurascentes, which now also includes C. mariae representing /Rapacea from the ITS phylogeny (FIG. 1). /Dermocybe s.l. is further supported as monophyletic group (BPP 92, BS 79). ML tree topologies consistently indicate a sister group relationship of /Der-mocybe s.l. and /Myxotelamonia.
When focusing on the phylogeny of classical subgenera in Cortinarius (Moser in Singer 1986), we further confirm that most subgenera of Cortinarius are polyphyletic (TABLE III); a monophyletic exception is subgenus Cortinarius. Species of subg. Dermocybe split into /Dermocybe s.l., /Icterinula and /Austronan-ceiensis, and monophyly of these three lineages cannot be excluded. Cortinarius species classified in subg. Leprocybe fall into the clades /Badiovinaceus, /Callisteus, /Leprocybe s.str., /Limonei, /Orellani and /Telamonia. Myxacium spp. form the lineages /Myxacium s.l., /Ochroleuci, and /Delibuti. Cortinarii classified in subg. Phlegmacium fall in the lineages /Calochroi, /Phlegmacium, /Purpurascentes, and /Myxotelamonia. Cortinarius species classified in subg. Sericeocybe can be found in the clades /Telamonia and /Anomali. Telamonia species are found in /Telamonia, /Dermocybe s.l., /Myxotelamonia and /Acutus.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) offers advantages over other methods in its ability to use complex models of evolution and in its ease of interpretation and computational efficiency. Phylogenetic analyses of multigene data, estimation of divergence times, testing monophyly of clades, inferring the root of a phylogenetic tree, mapping mutations on phylogenies, and evaluation of tree topologies have been addressed using Bayesian inference (Huelsenbeck et al 2002; Marvaldi et al 2002; Miller et al 2002; Nielsen 2002; Thorne and Kishino 2002; Garnica et al 2003a, b). In our datasets, Bayesian phylogenetic inference performed as well or better than ML. Bayesian inference is more practical and much faster than ML, especially when analyzing large datasets. BS values calculated using maximum parsimony or ML are generally lower than the corresponding BPP, especially in larger clades (Karol et al 2001, Murphy et al 2001, Huelsenbeck et al 2002, Miller et al 2002). The Bayesian method was evaluated critically and tested by computer simulations in two recent studies. Suzuki et al (2002), using three sets of four nucleotide sequences, showed that BPP can be excessively liberal when concatenated gene sequences are used whereas BS probabilities are slightly conservative. Wilcox et al (2002), using a ML model tree of 23 species of snakes, evaluated the reliability of BPP and BS as a statistical confidence of interior branches. They showed that as BPP underestimates less than BS, BPP is a better indicator of statistical confidence.
First consequences: the departure from old genus concepts. –
If taxonomic recognition is granted only to clearly monophyletic groups, all taxa except the Hebeloma outgroup must be regarded as members of one large genus. Thus, Cortinarius is an extremely ramified lineage of ectomycorrhizal mushrooms with a broad range of morphological variability. The genus circumscription had to be widened based on phylogenetic and morphological data only recently; Peintner et al (2001, 2002) clearly demonstrated that representative species of Thaxterogaster, Protoglossum and Quadrispora are reduced forms of Cortinarius. Therefore their generic names were synonymized under Cortinarius (Peintner et al 2002a, Kuhnert and Peintner 2003). Dermocybe, Rozites, Cuphocybe and Rapacea had been segregated from Cortinarius based on veil characteristics and chemotaxonomy (Heim 1951, Moser 1972, Singer 1986, Horak 1999). But molecular data (Liu et al 1997, Høiland and Holst-Jensen 2000, Seidl 2000, Peintner et al 2001, Peintner et al 2002c) suggested that some of these diagnostically valuable characters had been overweighted. The genus Dermocybe was delimited by the presence of anthraquinonic pigments, once regarded as restricted to this "genus" (Moser 1972, Singer 1986). But Gill and Steglich (1987) also recorded anthraquinones for some Telamonia and Phlegmacium species. Our current phylogenetic analyses show that Dermocybe taxa from both the Northern and Southern hemispheres nest within Cortinarius, supporting Dermocybe as a synonym of Cortinarius. The nesting of Rozites, Cuphocybe and Rapacea spp. also supports synonymy of those genera with Cortinarius (Peintner et al 2002c). The nomenclatural consequences proposed by Peintner et al (2002b) were based on both morphological and molecular data. Thus, the genus Cortinarius encompasses a morphologically highly variable group of mushrooms that includes both agaric (Cortinarius, Dermocybe, Rozites, Cuphocybe and Rapacea) and sequestrate (Thaxterogaster, Protoglossum, Hymenogaster pro parte and Quadrispora) forms. All these taxa, however, do share the typical basidiospore morphology and ectomycorrhizal strategy.
Toward a phylogeny-based taxonomy of Cortinarius. –
Subgenera are exceedingly helpful to subdivide large genera into smaller taxonomic units; they should be practical and reflect the hierarchy of evolutionary relationships. Phylogenetic research repeatedly has rejected the monophyly of Dermocybe, Leprocybe, Myxacium, Phlegmacium, Sericeocybe and Telamonia (Liu et al 1997, Chambers et al 1999, Høiland and Holst-Jensen 2000, Seidl 2000, Peintner et al 2001, Moser and Peintner 2002, Peintner et al 2002c, Garnica et al 2003b). Dermocybe and Telamonia both are paraphyletic and need to be redefined. The morphological and chemotaxonomical characters used to define Leprocybe and Sericeocybe (fluorescent substances or silky to glimmering pilei) appear to have been overweighted, thus creating artificial subgenera. The subgenera Myxacium and Phlegmacium, previously delimited solely by veil characters, probably deserve subdivision into smaller, monophyletic entities. All these smaller and/or partly new subgenera should be defined using both morphological and molecular data. Chemotaxonomy alone, while useful in delimiting species or sections, has not proven reliable for segregating subgenera (e.g., Dermocybe, Leprocybe). Delimitation of groups based on only a few morphological characters likewise has proven untrustworthy (e.g., Rozites, Phlegmacium, Myxacium).
The existing taxonomy needs to be re-evaluated to reflect an accurate phylogeny of Cortinarius. We base our Cortinarius phylogeny on the results of the Bayesian analysis of the two datasets (FIGS. 1, 2). Because the shortness of the basal branches greatly complicates generation of a Cortinarius phylogeny, for now, we offer a synthesis of the two Bayesian consensus trees as our best hypothesis.
Several larger monophyletic groups can be distinguished within Cortinarius (e.g., /Myxacium s.l., telamonioid group, /Dermocybe s.l., /Calochroi; FIGS. 1, 2), but our results do not support a split of Cortinarius into two distinct lineages as suggested by Høiland and Holst-Jensen (2000). The many lineages uncovered indicate that this species-rich genus requires more subgenera than are defined currently. Each of the well-supported clades discussed below may merit the rank of a subgenus, but we were unable to elucidate evolutionary relationships among these Cortinarius subclades. It is possible that the still comparatively small sample size is fundamentally responsible for this lack of resolution. This in turn suggests the need for combining morphological and molecular studies and including more species on a global scale before proposing any major taxonomic changes. Below we present the potentially diagnostic characters for each well-supported lineage revealed in our analyses.
The three distinct lineages of Myxacium species..
Cortinarius subg.
Myxacium is polyphyletic (Høiland and Holst Jensen 2000, Peintner et al 2001, Seidl 2000). Because C. collinitus is the type species of the subgenus, subg. Myxacium should be restricted to taxa of /Myxacium s.l. Our results indicate that the Ochroleuci and Delibuti, previously placed in Myxacium, derived from different lineages of Phegmacium spp. and thus are not closely related to /Myxacium s.l. More extensive taxon sampling is urgently needed to understand the evolution of the striking, strongly developed, viscid veils in Cortinarius.
Clade /Myxacium s.l.: Seidl (2000) already has shown that two sections of subg. Myxacium, sec. Myxacium and sec. Defibulati, form a monophyletic group with two distinct lineages. Our phylogenetic analysis has added species from the southern hemisphere. Cortinarius cycneus and "C. magellanicus" form a distinct lineage within /Myxacium s.l. This "C. magellanicus" from New Zealand is not conspecific with the material from the type locality in South America. More data from the numerous Southern Hemisphere Myxacium and Paramyxacium species are needed to determine whether Northern Hemisphere species evolved as a separate lineage due to geographic isolation.
Clade /Ochroleuci: Konrad and Maublanc (1948) established subg. Myxacium sec. Ochroleuci, based on the basidiome color and the bitter taste of the slimy veil. Bayesian posterior probabilities support sister group relationships of this lineage to /Allutus and /Calochroi, suggesting that viscid universal veils are derived secondarily in /Ochroleuci.
Clade /Delibuti comprises Northern and Southern Hemisphere species. Therefore it might be regarded as a comparatively old lineage. Horak and Wood (1990) hypothesized that C. salor and C. rotundisporus represent living fossils because they appear to have been ectomycorrhizal partners in the Nothofagus and Eucalyptus-Leptospermum forests in Gondwanaland millions of years ago. Relationships of C. iodes and C. salor, both classified in sec. Delibuti, remain unresolved. Common morphological characters are the bluish basidiome colors and (sub)globose basidiospores. However, as indicated by the numerous taxonomic shifts, there is considerable uncertainty regarding the systematic placement of many so-called Delibuti; C. australiensis has synonyms in the form genus Rozites, and C. rotundisporus also has been placed in Phlegmacium sec. Amarescentes. The combined analysis shows C. iodes and C. rotundisporus as sister groups, indicating a close relationship of /Delibuti to /Phlegmacium.
The lineages of Phlegmacium spp..
Our results and other recent molecular studies on Phlegmacium species (Moser and Peintner 2002a; Garnica et al 2003a, b) underscore three urgent reasons for revising traditional concepts of subg. Phlegmacium based on morphological, molecular and chemotaxonomical data: (i) subg. Phlegmacium is polyphyletic, (ii) many sections of Phlegmacium as defined by Moser (in Singer 1986) (e.g., Fulvi) are also polyphyletic; (iii) many clades implied by molecular analyses are related to other classically based taxa.
Using chemotaxonomical data, Brandrud (1998, 2002) proposed that section Fulvi is polyphyletic despite its apparent morphological homogeneity. Phlegmacium species with the most primitive biosynthetic pigment pathways also have high pigment concentrations, high pigment diversity and easily oxidized compounds. These supposed primitive pigment characters co-occur with such extreme "phlegmacioid" feature as bright coloration, strongly glutinous pileus surfaces, marginate bulbous stipe bases, citriform, coarsely verrucose spores, and absence of brown encrusted parietal pigments. Derived species are thought to possess more inconspicuous telamonioid characters, such as brown coloration, and less bulbous stipes (Brandrud 2002). We likewise observe two lineages with viscid universal veils derived from Cortinarius species producing phlegmacioid basidiomes. It would appear that the evolutionary trend for Phlegmacium spp. and perhaps Cortinarius in general is a decrease in pigment diversity and a shift away from spectacular toward less conspicuous basidiomes. Another observed trend is from complex agaricoid to reduced sequestrate fruit bodies (Peintner et al 2001).
The /calochroid group is a common lineage for Phlegmacium spp. and /Ochroleuci in subg. Myxacium. Garnica et al (2003a) also reported species from different sections of European Phlegmacium in /Calochroi. Morphological characters common to this group are citriform basidiospores, a well-developed gelatinous layer and epicutis, a simplex pileipellis, brightly colored basidiomes and a marginated bulbous stipe.
In addition, /Phlegmacium circumscribes taxa assigned to different sections of subg. Phlegmacium. Moreover, transitions between "classical" Phlegmacium and Myxacium spp. morphotypes can be observed also in this lineage; Cortinarius coerulescens has the blue colors typical for representatives of the species complex around C. iodes (subg. Myxacium) but with a less-developed viscid veil. C. iodes shows a sister group relationships with C. rotundisporus in the combined analysis, a representative of /Delibuti.
The lineage subg..
Cortinarius.
Representative species of subg. Cortinarius, one of the smallest subgenera with only ~10 species worldwide, form a well-supported lineage. Our results support segregation of small groups based on classical morphological concepts; subg. Cortinarius, as delimited from other Cortinarius subclades, produces large, fleshy basidiomes with dry, squamose-tomentose pileus surfaces, lamellae bearing cheilo- and pleurocystidia and tissues that turn red in KOH (Moser in Singer 1986).
Lineages of species with well-developed universal veils..
Basal relationships are not resolved for lineages /Rozites, /Cuphocybe, and /Achrous. These clades represent former Rozites or Cuphocybe taxa. Rozites has been inferred to be polyphyletic (Peintner et al 2002c). The amyloid reaction in the trama is the most conspicuous shared morphological character of species of /Rozites. Data including more taxa with amyloid trama reaction support a subg. Rozites circumscribing taxa around the type species, C. caperatus (Peintner unpublished).
The lineage(s) of Dermocybe..
The included Der-mocybe taxa fall into three lineages: /Dermocybe s.l., /Icterinula and /Austronanceiensis. Our data do not reject monophyly of these three clades. Important implications resulting from our phylogenetic analyses are: (i) all Dermocybe spp. analyzed are nested in Cortinarius, suggesting for Cortinarius systematics that they and probably all other Dermocybe species should bear a Cortinarius name; (ii) the lineage /Dermocybe s.l. can be considered as Cortinarius subg. Dermocybe (Fr.) Trog because this clade of Northern Hemisphere taxa includes the type of Dermocybe, C. cinnamomeus (L. : Fr.) Wünsche (Liu et al 1997); (iii) Northern Hemisphere taxa of /Dermocybe s.str. (sec. Dermocybe) appear derived from Southern Hemisphere taxa; (iv) /Dermocybe s.l. is paraphyletic, including taxa of subgenera Cystogenes and Telamonia. More species from the Southern Hemisphere should be sampled to test monophyly of Dermocybe and to elucidate the origin and evolution of this interesting group of fungi.
Clade /Dermocybe s.l. includes the subclades /Dermocybe s.str., /Splendida (D. splendida, C. globuliformis) as well as C. austrovenetus, C. firmus (Telamonia), C. olivaceopictus, and C. teraturgus (Cystogenes). A wide range of pigments can be found in taxa of this clade. Taxa of /Dermocybe s.str. contain dermorubin and sometimes also dermocybin (Keller 1982). C. olivaceopictus represents a separate lineage (Liu et al 1997). This corresponds with results of pigment analyses that showed that C. olivaceofuscus, which is related closely to C. olivaceopictus, to contain endocrocin and flavomannin-6,6-dimethylether but no dermorubin (Gruber 1975). Høiland (1983) regarded the C. olivaceopictus complex basal to Northern Hemisphere Dermocybe species because its pigment spectrum is less complex than found in /Dermocybe s.str. Pigments of C. firmus have not been investigated. Moser and Horak (1975) placed C. austrovenetus in Dermocybe subg. Icterinula, sec. Pauperae. Taxa of stirps Austroveneta typically have olivaceous colors. The two major pigments are the orange anthraquinone skyrin and the green-yellow pre-anthraquinone austrovenetin. Austrovenetin is unstable and on exposure to oxygen and sunlight changes chemically to protohypericin and the purple extended quinone hypercin (Gill 1995b). These pigments are related chemically to pigments in /Purpurascentes (sec. Scauri), atrovirin-B, skyrin, and (probably) hypericin (Keller et al 1987, Gill 1995a). Because D. splendida contains the rare tetrahydroanthraquinones, Gill et al (1995b) assume an isolated position for this taxon, suggesting a possible relationship to C. umbonatus and D. erythrocephala. Moser and Horak (1975) integrated C. teraturgus in subg. Cystogenes stirps Austrolimonius. Most species in Cystogenes have cheilocystidia, and orange to red-brown colors characterize species of stirps Austrolimonius. Chromatography demonstrated the lack of typical Dermocybe pigments (Gruber 1975), which is in agreement with the isolated position of C. teraturgus in /Dermocybe s.l.
Clade /Icterinula includes Southern Hemisphere species in Dermocybe subg. Icterinula, sec. Holoxantha, stirps Amoena. C. icterinus and C. amoenus have predominantly yellow pigments (endocrocin, dermolutein) and a slightly gelatinized pileipellis (Moser and Horak 1975, Garnica 2003b). Endocrocin and dermolutein are the principal anthraquinonic pigments (Gruber 1975).
Clade /Austronanceiensis includes Southern Hemisphere species in Dermocybe subg. Icterinula sec. Holoxantha stirps Nothoveneta. C. austronanceiensis differs from species in stirps Amoena by more robust basidiomes and pileipelli that are not gelatinize, but the main pigments of C. austronanceiensis are also endocrocin and dermolutein. Dermocybe cardinalis combines yellow, red and purple pigments that are produced by the cardinals, a series of unique naphthoquinone dimers. Because cardinals are unique for this species, Gill (1995b) proposed an isolated position for D. cardinalis. However, our phylogenetic analyses suggest a close relationship to Dermocybe austronanceiensis, providing further support for monophyly of /Icterinula and /Austronanceiensis.
The lineages of Telamonia spp.. The /telamonioid group is a lineage of /Telamonia /Orellani, /Anomali, and C. laetus. /Telamonia morphologically circumscribes a homogenous group of Cortinarii. Hygrophanous pilei, the lack of viscid or gelatinous veils and well-developed cortinas characterize most species. The fluid transitions among inconspicuous morphological characters make the systematics of this group particularly difficult. Compared to the other Cortinarius spp., members of /Telamonia all have ITS sequences with a ~50 bp gap in the ITS1 region. Moreover, there is a low divergence of ITS sequences in this group that makes it particularly difficult to resolve subclades within /Telamonia. More variable molecular regions have to be tested to infer for a meaningful phylogeny for this difficult group of "small brown mushrooms".
Species of /Orellani share many characters with species of /Telamonia. In addition, they contain bright blue or blue-green fluorescent substances and orellanine, a lethal mushroom nephrotoxin (Danel et al 2001). The placement of /Anomali in the telamonioid group also is in agreement with morphological characters typical for this group (subg. Sericeocybe sec. Anomali), e.g., slender basidiomes that lack viscid veils. Many species of sec. Sericeocybe in subg. Sericeocybe appear in /Telamonia. Such a taxonomic placement already was proposed by Brandrud et al (1995) based on morphological characters. Cortinarius laetus is a Telamonia spp. characterized by a conspicuous yellow universal veil and small, mycenoid aspect. Therefore, it was a surprise at first to discover that the morphologically similar species of /Acutus are a segregate of /Telamonia, as already shown by Høiland and Holst-Jensen (2000). Peintner et al (2003) more recently have demonstrated that at least five species (including one sequestrate) belong to this lineage. On closer examination, species of /Acuti differ from other Telamonia spp. by the typical structure of the lamellar trama consisting of ellipsoid inflated hyphae and by the presence of numerous cheilocystidia. The independent origin inferred for /Acutus suggests that the mycenoid habit originated at least twice in Cortinarius: once in the /telamonioid group (C. pulchellus, C. laetus) and once in /Acutus.
The lineages of Leprocybe spp..
The subg.
Leprocybe as currently recognized is polyphyletic: some clades or species cluster in the /telamonioid group (/Orellani, C. bolaris, C. raphanoides, C. gentilis), others form lineages with unknown relationship (/Callis-teus, /Limonius, /Leprocybe). The fluorescent pigments that define this subgenus do not appear to be synapomorphic. Tree topologies indicate a relationship of /Callisteus and /Limonius to Phlegmacium species, from which they mainly differ by the reduction of the viscid pileipellis. More extensive sampling will prove our hypothesis that this subgenus has to be restricted to /Leprocybe as typified by C. cotoneus.
Can the Cortinarius phylogeny be resolved? –
Our results reveal many fundamental problems for Cortinarius systematics. First, it is difficult to identify and delimit taxa reliably within this species-rich genus, especially taxa from different geographical regions. "C. magellanicus" from New Zealand (/Cycneus), for instance, is not conspecific with (and in fact well separated from) C. magellanicus (/Ochroleuci) from the type locality in South America (FIG. 1). For reliable phylogenetic data it often is necessary to analyze sequences from at least two or more collections of one species and if possible from different geographical origins (Peintner et al 2001, Moser and Peintner 2002a, b, Peintner et al 2002c, Garnica et al 2003b).
Resolution and support in a phylogeny can be improved by increasing the number of characters (Soltis et al 1998, 1999). Here, character sampling helped to resolve relationships between (sub)clades of Cortinarius. In general, increased character sampling increased BPP for larger clades showing low support (/Dermocybe s.l.), or recovered and supported new, larger clades (e.g., the telamonioid group).
On the other hand, increased taxon sampling resolved relationships in terminal branches of the phylogeny, e.g., within clades. The lineages /Purpuras-centes and /Phlegmacium were not supported or only weakly supported in the combined analysis (FIG. 2), yet a significantly higher BPP was obtained with increased taxon sampling (FIG. 1). This indicates that a minimum number of representative species must be sampled to recover a clade in multigene phylogenies. Extensive taxon sampling still is regarded as one of the most reliable ways to increase overall phylogenetic accuracy (Hillis 1996, 1998, Pollock and Bruno 2000, Pollock et al 2002, Zwickl and Hillis 2002). Thus, in addition to the low divergence in rDNA sequences, insufficient taxon sampling can confuse Cortinarius phylogeny. Increased taxon sampling has helped to resolve the phylogeny of closely related Cortinarius species (Liu et al 1997, Seidl 2000, Moser and Peintner 2002, Garnica et al 2003, Peintner et al 2003). Cortinarius, one of the most species-rich ectomycorrhizal mushrooms genera, has a worldwide distribution, yet most subgenera and sections are typified by species from the Northern Hemisphere. Selecting the type species for analysis was appropriate, but in so doing we strongly biased our results toward the Northern Hemisphere. Consequently, future research should include more species from the Southern Hemisphere. A meaningful phylogeny of this morphologically plastic and large genus cannot be based on a mere 5–10% of the known species. It would be appropriate to focus future phylogenetic analyses first on single clades and to sample taxa from both hemispheres to better understand the evolution of Cortinarius and thus to resolve, step by step, the challenging taxonomic problems of this fascinating genus.
The insights gained by a thorough investigation on Cortinarius phylogeny and evolution can be related to other fungal groups, especially ectomycorrhizal basidiomycetes. Ectomycorrhizal fungi are concentrated in the bolete clade, thelephoroid clade, cantharelloid clade, gomphoid-phalloid clade and in the euagarics clade (Amanita, Hygrophorus, Tricholoma, In-ocybe, Cortinarius, Phaeocollybia, Hebeloma and Laccaria) (Hibbett and Thorn 2000). The existence of numerous, relatively large clades of ectomycorrhizal fungi indicates that this ecological habit has been relatively stable at least during the recent radiation of euagarics (Moncalvo et al 2000, 2002). The phylogeny of ectomycorrhizal basidiomycetes has been addressed in many studies (e.g., Binder 1999, Jarosch 2001, Grubisha et al 2002, bolete clade; Kõljalg et al 2000, 2002, thelephoroid clade; Dahlman et al 2000, cantharelloid clade; Humpert et al 2001, gomphoid-phalloid clade; Miller et al 2001, russuloid clade; Drehmel et al 1991, Amanita; Aanen et al 2000, Hebeloma) most of them using rDNA sequence data. Few studies furnish comparatively well-resolved phylogenies on different taxonomic levels (Drehmel et al 1991, Grubisha et al 2002, Miller 2003), but some subclades often remain unresolved, and the phylogenies of large genera are especially difficult to resolve (Binder 1999, Humpert et al 2001, Miller et al 2001). Strategies developed to resolve the phylogeny of Cortinarius and the resulting insights in the taxonomic value of morphological and ecological characters could help to address the phylogeny, systematics and evolution of many other groups of fungi.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: Ursula.peintner{at}uibk.ac.at
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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