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Phylogenetic relationships of European Phlegmacium species (Cortinarius, Agaricales)^*

     Lehrstuhl für Spezielle Botanik und Mykologie, Botanisches Institut, Universität Tübingen, Auf der Morgenstelle 1, D-72076 Tübingen, Germany

B. Oertel

     Institut für Gartenbauwissenschaft, Universität Bonn, Auf dem Hügel 6, D-53121 Bonn, Germany

F. Oberwinkler

     Lehrstuhl für Spezielle Botanik und Mykologie, Botanisches Institut, Universität Tübingen, Auf der Morgenstelle 1, D-72076 Tübingen, Germany

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

Phylogenetic relationships of 54 European Phlegmacium species, including members of most of the sections of classical systematics, were studied, integrating macro-, micromorphological and chemical characters of the basidiomes, as well as molecular phylogenetic analysis of nuclear rDNA sequences. Microscopical structures of the basidiomes were studied by light microscopy. Basidiospore morphology was examined by scanning electron microscopy. Internal-transcribed spacers (ITS 1 and 2, including the 5.8S) and the D1/D2 (LSU) regions of nuclear rDNA were sequenced and analyzed with a Bayesian Markov chain Monte Carlo approach. Many subgroups detected by the molecular analysis are related to groups known from classical systematical concepts. Among others, these subgroups were significantly supported: i) a group containing most of the members of section Fulvi ss. Brandrud and the species Cortinarius arcuatorum, C. dibaphus and C. multiformis; ii) a group comprising taxa of section Calochroi ss. Brandrud and the species C. fulvocitrinus and C. osmophorus; iii) a group containing species of section Glaucopodes ss. Brandrud and C. caerulescens; iv) a group including members of section Phlegmacioides ss. Brandrud; v) a group that includes the species C. cephalixus, C. nanceiensis and C. mussivus. Stipe shape, color of flesh, pigment contents, KOH reaction on pileipellis and gelatinous layer, degree of development of a gelatinous layer on the pileipellis, and pileipellis structure were useful characters in delimiting subgroups in Phlegmacium, while basidiospore morphology was significant at species level. With the exception of C. glaucopus, C. infractus and C. scaurus, ITS and D1/D2 sequences obtained from collections of the same species from different geographical origins showed very little variation. Our molecular and morphological analyses suggest revisions of the traditional concepts of the subgenus Phlegmacium in Europe.

Key words: D1/D2 domains, Europe, ITS, LSU, molecular phylogeny, morphology, nuc rDNA, systematics

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Phlegmacium Fr., a subgenus of Cortinarius, includes species with relatively fleshy basidiomes with vivid colors, a viscid to glutinous pileus surface and a dry stipe. Many Phlegmacium species have a wide distribution in Europe and occur in ectomycorrhizal association with coniferous and deciduous trees. Some species are supposed to be even more widespread and reach North America (Moser et al 1994, 1995; Moser and Ammirati 1996, 1997, 1999). On the other hand, other Phlegmacium species show a limited distribution, mainly due to high host-tree specificity, or specific climatic or edaphic requirements.

Since Fries (1821) introduced the name Phlegmacium as a tribus of the genus Agaricus, many mycologists have contributed to the systematics and taxonomy of the Phlegmacium species in Europe, grouping them in a separate genus (e.g., Wünsche 1877, Fayod 1889, Earle 1909, Ricken 1915, Moser 1960) or in a subgenus of Cortinarius (e.g., Fries 1836–1838, 1878–1884; Moser 1983; Moënne-Loccoz et al 1990–2001; Bidaud et al 1994; Brandrud et al 1990–1998). Modern classification systems recognize Phlegmacium as a subgenus of Cortinarius. Species delimitations in Phlegmacium traditionally have been based almost exclusively on "field recognition", e.g., coloration of pileus and lamellae, as well as macrochemical tests combined with basidiospore morphology (e.g., Moser 1960, 1983, 1986; Moënne-Loccoz et al 1990–2001; Bidaud et al 1994). Attempts to increase the number of characters used for taxonomic purposes in Phlegmacium have been made by Oertel (1984), Oertel and Laber (1986), Steglich et al (1984), Steglich and Oertel (1985), Gill and Steglich (1987) and Brandrud (1998b); all of these authors included pigment chemistry of the basidiomes. Brandrud (1996a, b, 1998a) reported the taxonomic significance of microscopical characters referring to veil and pileipellis structure for the delimitation of some sections in Phlegmacium.

Various classification systems have been proposed for Phlegmacium in Europe. Moser (1960), who monographed most of the Phlegmacium species in this region, recognized these sections: Amarescentes, Caerulescentes, Calochroi, Fulvi, Laeticolores, Phlegmacium and Triumphantes. Moser (1983) defined a new section Tenues and changed the name of section Laeticolores to Scauri. Bidaud et al (1994) and Moënne-Loccoz et al (1990–2001) included some taxa from the subgenera Myxacium and Sericeocybe in Phlegmacium and recognized the sections Caerulescentes, Claricolores, Delibuti, Fulgentes, Glaucopodes, Laeticolores, Multiformes, Paties, Phlegmacium and Thalliophili. Brandrud et al (1990–1998) and Brandrud (1996a, b, 1998a, b) made several emendations of previous classification systems. They incorporated a large quantity of microscopic and chemical characters and used numerical analyses to split Phlegmacium into sections Caerulescentes, Calochroi, Elastici, Fulvi, Glaucopodes, Infracti, Multiformes, Phlegmacioides, Phlegmacium, Scauri and Subtorti. Molecular analyses based on the ITS (1 and 2) and 5.8S regions of nuclear rDNA recently have been applied to a limited number of European Phlegmacium species (Høiland and Holst-Jensen 2000, Peintner et al 2001).

Using original morphological and molecular data obtained from Friesian's and Henry's Phlegmacium species, we have tried to evaluate macro- and micromorphological as well as chemical characters, by comparing them through molecular phylogenetic analyses. Our species sampling includes members of most of the Phlegmacium sections recognized in traditional systematics. To get information on the degree of intraspecific variation of the molecular data, we mostly sequenced several collections of the same Phlegmacium species from different sites in Europe. Our phylogenetic hypotheses are discussed with current classification systems applied to Phlegmacium species in Europe.

	MATERIAL AND METHODS

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Material studied – A total of 86 collections, representing 54 European Phlegmacium species, were included in this study. Collection sites, host trees, locations of vouchers and GenBank accession numbers of the Phlegmacium species used in the morphological and molecular analyses are listed in Table I. We chose Laccaria amethystina Cooke, GenBank accession No. AF539737 (Garnica et al 2003) as outgroup species for the present molecular phylogenetic analysis, according to results obtained with D1/D2 sequences from a broad sampling of Agaricales (S. Garnica and M. Weiß unpubl) and multigene analyses by Binder and Hibbett (2002). Detailed macroscopical descriptions were made from fresh basidiomes and complemented with color photographs. Macrochemical tests with 40% KOH solution were performed on pileus surface, context and stipe base of the basidiomes. Identification of the collections were based on taxonomic keys of Moser (1960, 1983) and Brandrud (1996a, b, 1998a).

View larger version (78K): [in this window] [in a new window]   FIGS. 9–10. Pileipellis anatomy. 9. Longitudinal section of the pileus of Cortinarius calochrous (pileipellis simplex) showing gelatinous layer, epicutis, and context. 10. Longitudinal section of the pileus of C. viridocaeruleus (pileipellis duplex) showing viscid layer, epicutis, hypocutis, and context. Scale bar = 20 µm

View larger version (142K): [in this window] [in a new window]   FIGS. 11–18. Some principal basidiospore shapes in European Phlegmacium species. Ellipsoid. 11. Cortinarius scaurus. Ellipsoid-amygdaliform. 12. C. vulpinus. Amygdaliform. 13. C. cephalixus. Ellipsoid-amygdaliform. 14. C. purpurascens. Subglobose. 15. C. caesiocortinatus. 16. C. infractus. Citriform. 17. C. anserinus. 18. C. elegantior. Scale bars: 11–13 = 4 µm, 14 = 2 µm, 15 = 4 µm, 16 = 2 µm, 17–18 = 4 µm

An alignment of 86 sequences, representing 54 Phlegmacium species and Laccaria amethystina as outgroup species, was made with the MegAlign module of the Lasergene software system (DNASTAR Inc.), followed by manual adjustments in Se-Al (Rambaut 1996). Sequence alignments may be obtained from TreeBase (http://treebase.bio.buff-alo.edu/treebase/). Regions with ambiguous alignments, which occupied positions 1–60, 196–209, 236–249, 351–358, 537–567, 581–590, 599–613, 656–666, 705–724, 760–777 and 1412–1422 in our data matrix, were excluded for the phylogenetic analysis.

To estimate the phylogenetic relationships of the Phlegmacium species, the DNA alignment was analyzed using a Bayesian approach based on Markov chain Monte Carlo (MCMC; Larget and Simon 1999), as implemented in the computer program MrBayes 2.01 (Huelsenbeck and Ronquist 2001). In contrast to the maximum-likelihood method (Felsenstein 1981), in which the probability of the DNA alignment conditional on phylogenetic trees (the "likelihood" of the phylogenetic trees) is maximized, the Bayesian MCMC approach allows estimation of the a posteriori probability of phylogenetic trees, i.e., the probability that a tree is the true phylogenetic tree given the DNA alignment. Because the posterior probability distribution of the tree space is analytically inaccessible, the method uses a Monte Carlo technique to collect a large representative sample of phylogenetic trees from the tree space, from which the posterior probabilities can be estimated. By summing posterior probabilities of those trees in which a group of taxa is monophyletic it also is possible to estimate the a posteriori probability for the monophyly of given groups, i.e., the probability that a group is monophyletic given the DNA alignment. The power of this method to reconstruct phylogenetic relationships efficiently has been demonstrated by Murphy et al (2001) for mammalian phylogeny and by Maier et al (2003) and Garnica et al (2003) for several fungal groups. To improve mixing of the chain, we ran four incrementally heated simultaneous Monte Carlo Markov chains (Metropolis-coupling technique; see Huelsenbeck et al 2002) over 2 000 000 generations, using the general time-reversible model of DNA substitution with gamma-distributed substitution rates (see Swofford et al 1996), random starting trees and default starting parameters of the DNA substitution model. Trees were sampled every 100 generations, resulting in an overall sampling of 20 000 trees. From those trees that were sampled after the process had reached stationarity, a 50% majority-rule consensus tree was computed to get estimates for a posteriori probabilities. Branch lengths of this consensus tree were estimated with PAUP 4.0b10 (Swofford 2001) using maximum likelihood. The Bayesian MCMC phylogenetic analysis was repeated three times, always using random starting trees, on a Macintosh G4 computer to test the independency of the results from topological priors (Huelsenbeck et al 2002).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Macroscopical characters – Habit. Figures 1–8 show some selected basidiome habits of Phlegmacium species studied. Size of the basidiomes varied from medium to relatively large and robust. Basidiomes of Phlegmacium species collected on oligotrophic to mesotrophic soils were medium size (e.g., C. scaurus and C. subtortus), whereas the species from eutrophic-calciphilous soils were characterized by fleshy and relatively robust basidiomes.

View larger version (47K): [in this window] [in a new window]   FIGS. 1–8. Basidiome habits of some European Phlegmacium species. 1. Cortinarius multiformis. 2. C. elegantior. 3 C. vulpinus. 4. C. glaucopus. 5. C. saginus. 6. C. osmophorus. 7. C. scaurus. 8. C. praestans. Scale bar = 2 cm

Macroscopical characters – Stipe. The shape of the stipe base varied from attenuate to subradicating (C. turmalis, C. vulpinus), cylindrical to clavate (C. cephalixus, C. cumatilis, C. infractus, C. lustratus, C. coalescens, C. mussivus, C. porphyropus, C. saginus, C. scaurus, C. triumphans, C. variiformis, and C. varius) and bulbous (rounded, submarginate to marginate) in the remaining taxa studied (see Fig. 19). Scaly to floccose ring-like zones that become ocher-brown in age were found in the lower part of the stipe in C. triumphans, C. saginus and C. vulpinus. Ocher ring-like velum zones were observed in C. anserinus, C. varius and C. variiformis; these were brown in C. nanceiensis and slightly blue in C. praestans.

View larger version (41K): [in this window] [in a new window]   FIG. 19. Bayesian inference of phylogenetic relationships in European Phlegmacium species: Markov chain Monte Carlo analysis of an alignment of nuclear rDNA sequences from the ITS region (including the gene coding for the 5.8S ribosomal subunit) and the D1/D2 region of the large ribosomal subunit, with the general time-reversible model of DNA substitution with gamma-distributed substitution rates (GTR+G), random starting trees, default starting parameters of the substitution model and involving four incrementally heated Markov chains. Majority rule consensus tree from 18 000 trees sampled after convergence to stationarity; the topology was rooted with Laccaria amethystina. Numbers on branches are estimates for a posteriori probabilities. Branch lengths are maximum-likelihood estimates and are scaled in terms of expected numbers of nucleotide substitutions per site. Characters indicated; I) pileipellis duplex present () or absent (), II) basidiospore shape: a = amygdaliform, ac = amygdaliform to slightly citriform, c = citriform, e = ellipsoid, ea = ellipsoid-amygdaliform, s = subglobose and sf = subfusoid, III) bulbous stipe (submarginate to marginate) present () or absent (), IV) color of basidiome flesh (context) in fresh material: v = with violet shade, w = white (sometimes slightly ocher) and y = yellow to green, V) wine-red microscopical reaction with 3% KOH on pileus sections (and sometimes on lamellar trama) present () or absent ().

Macroscopical characters – Odor. The Phlegmacium species C. lustratus, C. dionysae and C. flavovirens are characterized by a distinctive farinaceous odor, C. osmophorus and C. odoratus by a sweet odor such as Citrus blossoms, C. odorifer by aniseed odor and C. mussivus by an apple-like odor later becoming unpleasant; an unpleasant odor reminiscent of rotten potatoes characterizes C. claroflavus and a soil-like odor C. variicolor.

Macroscopical characters – Macrochemical reaction. Many Phlegmacium species showed a distinctive color reaction with KOH on pileus surface, context and stipe base. The species C. boudieri, C. scaurus and C. flavovirens did not show any color reaction with KOH. The context and lamellae of C. scaurus, C. porphyropus and C. purpurascens turned purple when touched or scratched.

Microscopical characters – Gelatinous layer. In all Phlegmacia studied, the outer layer of the pileus was slightly gelatinous (viscid) to gelatinous. Hyphae of the gelatinous layer originate from the universal veil and the upper stratum of the epicutis. In C. variicolor, C. coalescens and C. balteatocumatilis, the poorly developed gelatinous layer was composed of relatively few hyphae; intermediate development of the gelatinous layer was observed in C. caerulescens, C. glaucopus, C. pseudonapus, C. saginus, C. triumphans, C. variiformis and C. varius, whereas in the remaining analyzed taxa the gelatinous layer was composed of various hyphal strata. The hyphae involved were relatively thin, cylindrical, hyaline to pigmented, sometimes weakly zebra-striped or with granular epiparietal incrustations and embedded in a matrix. The orientation of the hyphae in the deeper part was nearly parallel to the basal epicutis and becoming irregularly ascending toward the outer part. The hyphae of the universal veil on the pileus surface were narrow, hyaline to slightly pigmented.

Microscopical characters – Pileipellis. The distribution of this character in the studied Phlegmacium species is given in Fig. 19. We recognized two pileipellis types: a "pileipellis simplex" consisting only of an epicutis (Fig. 9) and a "pileipellis duplex" consisting of an external epicutis and an internal hypocutis (Fig. 10). Intermediate forms were observed in C. variicolor. The epicutis consisted of an upper zone, forming part of the gelatinous layer with subparallel to ascendent sparse hyphae, and a basal zone, formed by densely arranged hyphae in subradial arrangement. In older basidiomes, the hyphae of the basal part of the epicutis sometimes become faintly striped or pigmented with punctate epiparietal incrustations. The hypocutis was characterized by ovoid, ellipsoid to subglobose, hyaline to pigmented hyphal segments, which sometimes were embedded in a colored amber-like matrix.

Microscopical characters – Context. The pileus context consists of hyaline to pigmented, narrow or inflated hyphae. Inflated hyphae were predominant toward the lower layers of the context. In addition, cylindrical to tortuous oleiferous hyphae were observed in the pileus context.

Microscopical characters – Lamellar structure. The lamellar trama was regular, consisting of a lateral stratum of long, cylindrical, relatively narrow, parallel to subparallel, hyaline to pigmented hyphae, and a mediostratum with long, cylindrical to relatively short and inflated hyphal segments in parallel arrangement, especially toward the pileus context. Old basidia with wine-red content were present in C. cereifolius, C. claroflavus, C. elegantior, C. flavovirens, C. fulvocitrinus, C. nanceiensis, C. mussivus, C. splendens and C. meinhardii; basidia with purple content were found in C. cupreorufus, C. odorifer, C. prasinus, C. rufoolivaceus and C. xanthophyllus. Lageniform pleuro- and cheilocystidia, with epiparietal incrustations in fresh material characterize C. subtortus. Abundant cylindrical to tortuous sterile hyphal elements were observed on the lamellar edges of C. arcuatorum, C. dibaphus and C. prasinus.

Microscopical characters – Basidiospores. Figures 11–18 show the main types of basidiospore shape in the studied Phlegmacium spp.; an overview is given in Fig. 19. Basidiospores most frequently were citriform, followed by amygdaliform shape; few taxa possessed subfusoid or subglobose basidiospores. An infraspecific variation of the basidiospores ranging from amygdaliform to slightly citriform (= subcitriform) was found in various taxa. Most Phlegmacia studied were characterized by moderately to strongly ornamented basidiospores, with the exception of C. cumatilis, C. lustratus and C. turmalis, in which the basidiospores were scarcely ornamented.

Microscopical characters – Hyphal coloration in 3% KOH. Most external hyphae of the gelatinous layer turned dark-olive with a gray tone when treated with 3% KOH in C. atrovirens, C. ionochlorus, C. odoratus, C. splendens and C. meinhardii, whereas they became yellowish-green in C. cephalixus, C. nanceiensis, C. mussivus, C. prasinus, C. scaurus and C. xanthophyllus and pink in C. arcuatorum and C. sodagnitus. Yellowish-golden to yellowish-brown coloration of the epicutis and hypocutis characterized C. balteatocumatilis, C. caerulescens, C. cephalixus, C. coalescens, C. pseudonapus, C. saginus, C. triumphans, C. variicolor, C. variiformis, C. varius and C. vulpinus. In C. arcuatorum and C. dibaphus, the epicutis and context turned pink, and in various taxa a wine-red reaction was observed (see Fig. 19).

Microscopical characters – Phylogenetic analysis. Results from the Bayesian molecular phylogenetic analysis are illustrated in Fig. 19. The groupings obtained from phylogenetic analysis showed many correlations with current classification concepts for species of Phlegmacium in Europe (Moser 1960, 1983, 1986; Brandrud et al 1990–1998; Bidaud et al 1994; Brandrud 1996a, b, 1998a; Moënne-Loccoz et al 1990–2001). The sections proposed by Brandrud et al (1990–1998) and Brandrud (1996a, b, 1998a) show an especially high degree of congruence with subgroups detected in our analyses. Among others, these subgroups are supported by our molecular analysis:

The species assigned to section Fulvi were distributed in two clusters. The main cluster (Fig. 19, top), containing most species of section Fulvi, was divided into two subgroups; one of these is formed by the species C. arcuatorum, C. dibaphus, C. pseudofulmineus, C. elegantior, C. meinhardii, C. odoratus, C. splendens, C. ionochlorus, C. atrovirens and C. cereifolius. The species C. arcuatorum and C. dibaphus (the latter included in section Calochroi in the Brandrud system) consistently were placed within section Fulvi as neighbors to C. pseudofulmineus. Molecular analysis significantly supported subsection Atrovirentes within section Fulvi, containing the species C. odoratus, C. ionochlorus and C. atrovirens. The species pair C. meinhardii and C. splendens were included in this subsection. The remaining species belonging to the Fulvi main group, C. citrinus, C. xanthophyllus, C. prasinus, C. multiformis ss. M. M. Moser, C. rufoolivaceus, C. odorifer, C. cupreorufus, C. flavovirens and C. claroflavus appear basal to those discussed above; their phylogenetic relationships were unresolved in our analysis. A second cluster, including the species C. nanceiensis and C. mussivus, was placed outside the Fulvi main group, close to C. cephalixus. The species C. fulvocitrinus was placed in section Calochroi.

Section Calochroi, including the species C. calochrous, C. calochrous var. coniferarum, C. sodagnitus and C. citrinolilacinus. The species C. osmophorus and C. fulvocitrinus also were assigned to this group in our analysis.

Section Glaucopodes, containing the species C. anserinus, C. dionysae and C. glaucopus. The taxa C. viridocaeruleus and C. caerulescens also were included in this section.

Section Phlegmacium containing the species C. saginus, C. vulpinus, C. varius, C. triumphans and C. variiformis. The species C. saginus and C. vulpinus were significantly grouped together.

Section Phlegmacioides, including the species C. variicolor, C. coalescens and C. balteatocumatilis; the species C. coalescens and C. variicolor were grouped together.

Section Scauri, including the species C. scaurus, C. porphyropus and C. purpurascens; C. porphyropus and C. purpurascens appear as sister taxa.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
In this section we discuss our results, comparing our molecular phylogenetic hypotheses with the distribution of pigment contents and macro- and microscopical characters of the basidiomes. According to the congruence of our molecular phylogenetic results and the classification system proposed by Brandrud et al (1990–1998) and Brandrud (1996a, b, 1998a) for European Phlegmacia, we will base our remarks on this classification concept, although various species examined in this study have not yet been included in this system (see Table II).

Section Fulvi – The main cluster containing most of the species of this section was supported by an a posteriori probability value of 80%, containing an even better-supported subgroup (100% a posteriori probability) that includes the species C. arcuatorum, C. dibaphus, C. pseudofulmineus, C. elegantior, C. meinhardii, C. odoratus, C. splendens, C. ionochlorus, C. atrovirens and C. cereifolius (Fig. 19, top). The species C. arcuatorum and C. dibaphus, which are considered closely related in current classification systems (Moser 1960, 1983, 1986; Moënne-Loccoz et al 1990–2001), are linked with an a posteriori probability of 100%. According to our results, C. provencalis occupies an isolated position and is placed basally to sections Fulvi and Calochroi.

Our phylogenetic analysis supports a close phylogenetic relationship between subsection Atrovirentes (C. odoratus, C. ionochlorus and C. atrovirens) and subsection Splendentes (C. meinhardii and C. splendens). A close relationship between C. odoratus, C. ionochlorus and C. atrovirens grouped in subsection Atrovirentes first was proposed by Oertel (1984) and Oertel and Steglich (1985), who detected atrovirin and flavomannin as main pigments, while a 4-hydroxy-flavomannin-6,6'-dimethylether pigment was detected in C. meinhardii and C. splendens (subsection Splendentes). Moreover, we have observed a similar KOH reaction of the outermost external hyphae of the gelatinous layer in the members of both subsections. A distinctive character of members of subsection Atrovirentes is that the basidiomes become black pigmented when dried (Steglich and Oertel 1985). The specimens of C. ionochlorus and C. atrovirens, traditionally distinguished by the coloration of the lamellae and ecology, showed identical DNA sequences. The differences in the lamellar coloration probably are caused by light-sensitive pigments under different ecological conditions: Basidiomes of C. ionochlorus, which are associated with frondose trees, grow under a layer of fallen leaves in their early developmental stage and thus are shielded from sunlight. Conversely, the basidiomes of C. atrovirens, which are affected early by sunlight, thus might become strongly pigmented.

Phylogenetic relationships among C. citrinus, C. xanthophyllus, C. prasinus, C. multiformis, C. rufoolivaceus, C. odorifer, C. cupreorufus, C. flavovirens and C. claroflavus remained unresolved in our molecular phylogenetic analysis due to very similar sequences in the studied rDNA regions. This is congruent with the uniformity of microscopic structures that characterize these species. Cortinarius nanceiensis and C. mussivus were placed together but separate from the remaining members of section Fulvi. Both taxa are well characterized by a cylindrical to clavate stipe, a character not present in the remaining taxa of section Fulvi that we examined. Similarities in pigment contents—phlegmacin-8'-methylether, which has been considered as a derived character in this group (Oertel 1984, Steglich and Oertel 1985, Brandrud 1998b)—support the close relationship between C. nanceiensis and C. mussivus. These two species clustered with C. cephalixus in our molecular phylogenetic analysis (95% a posteriori probability), which is consistent with similarities in stipe shape, pileipellis structure and probably in pigments of the basidiomes. Cortinarius fulvocitrinus, which contains flavomannin-6,6'-dimethylether (Oertel 1984, as C. citrinus), was classified in section Fulvi and subsection Splendentes by Brandrud (1998b). However, unlike other members of section Fulvi, the lamellae of C. fulvocitrinus are not yellow. Consistent with this observation, C. fulvocitrinus was separated from section Fulvi and placed among members of section Calochroi in our molecular analysis.

Section Calochroi – The grouping of C. calochrous, C. calochrous var. coniferarum, C. sodagnitus, C. citrinolilacinus, C. osmophorus and C. fulvocitrinus was supported by an a posteriori probability of 95%. The species C. osmophorus was ascribed to section Triumphans by Moser (1960, 1983, 1986), and C. fulvocitrinus was placed in section Fulvi (Brandrud 1998b; see discussion above). Similarities in habit concerning the stipe shape, flesh coloration (except C. fulvocitrinus), pilleipellis structure (pileipellis simplex), degree of development of the gelatinous layer and basidiospore morphology (except C. sodagnitus) can be correlated with our molecular grouping. While yellow to yellowish-brown colors of the pileus characterize all the species in this section, further studies of pigment contents might help to clarify the delimitation of this group. The analyzed sequences of C. calochrous and C. citrinolilacinus were identical, reflecting the poor morphological differences on which the separation of both species is based (Moser 1960).

Section Glaucopodes – Section Glaucopodes, containing the species C. anserinus, C. dionysae, C. glaucopus with the incorporation of C. viridocaeruleus and C. caerulescens, was supported as a monophyletic group by an a posteriori probability of 96%. In classical systems (Moser 1960, 1983, 1986; Brandrud et al 1990–1998; Moënne-Loccoz et al 1990–2001), the latter two species were classified in different sections (Table II). Macroscopical characters that correlate with the grouping in our analysis are similarities of basidiome coloration and stipe shape. At the microscopic level, all members in this group present a similar pileipellis structure with a moderately developed gelatinous layer. The grouping of C. viridocaeruleus with C. anserinus and C. dionysae, which is present in our molecular analysis, is consistent with the basidiospore morphology of these species.

Section Phlegmacium – The species C. saginus, C. vulpinus, C. varius, C. triumphans and C. variiformis were classified by Brandrud (1996a, b) in section Phlegmacium, which is supported by an a posteriori probability of 64% in our molecular analysis. Different systematic positions of these species have been proposed in the past (Moser 1960, 1983, 1986; Moënne-Loccoz et al 1990–2001). Members of section Phlegmacium are characterized by similarities of pileus and lamellar coloration, by a reduced gelatinous layer and by stipe shape. The presence of a veil in C. saginus, C. triumphans and C. vulpinus has been suggested as an adaptation to xerophilic conditions by Brandrud (1996a, b). At the microscopic level, the pileipellis morphology characterizes this group well; a similar observation was reported by Brandrud (1996a, b). Cortinarius saginus and C. vulpinus were grouped together with an a posteriori probability of 85% in our molecular analysis; the phylogenetic relationships among the other members of this section remained unresolved.

Section Phlegmacioides – The species of section Phlegmacioides (C. variicolor, C. coalescens and C. balteatocumatilis) included in our study were clustered together with an a posteriori probability of 92%. In traditional classification systems these species have been considered closely related (Moser 1960, 1983, 1986; Brandrud et al 1990–1998; Brandrud 1998a; Moënne-Loccoz et al 1990–2001). Macromorphological traits consistent with this association are the texture and consistency of the pileus surface, the stipe shape and basidiome coloration. The violet pigments turn brown with the age of the basidiomes (Brandrud 1998a). The flesh becomes yellow with KOH. At the microscopic level, members of this section have a poorly developed gelatinous layer and hypocutis. Though grouped together with an a posteriori probability of 100%, C. variicolor and C. coalescens appear to represent different species according to their genetic distance. Brandrud (1998a) differentiated them by ecology, odor and coloration of the basidiomes.

Section Multiformes – The close phylogenetic relationship between C. praestans and C. cumatilis was strongly supported by an a posteriori probability of 98% in our molecular analysis. This agrees with classical taxonomy, in which both species also have been treated as closely related. The basidiomes of both species show similar coloration and differ in their basidiospore morphology. In our molecular analysis, C. multiformis ss. M.M. Moser was separated from these species and grouped among members of section Fulvi; this is justified because of stipe shape and probably of basidiome pigments. There is some taxonomic confusion regarding the latter taxon: It is possible that C. multiformis ss. Brandrud, which is not included in the present molecular study and which differs from C. multiformis ss. M.M. Moser in habit and ecology, represents a different species that is phylogenetically closer to C. praestans and C. cumatilis.

Section Infracti – The DNA sequences of three collections of C. infractus showed a certain variability. In tree topology, C. infractus is related to C. subtortus, with an a posteriori probability of 96%. However, C. subtortus also was placed in the group with C. cephalixus, C. nanceiensis and C. mussivus in some re-iterations of the MCMC analysis (data not shown). Moser (1960, 1983, 1986) included C. subtortus and C. infractus in section Amarescentes, based on their lamellar coloration, a bitter taste and basidiospore morphology. Cortinarius subtortus is distinguished by the presence of cystidia and C. infractus by indole alkaloid content (Steglich et al 1984).

Section Scauri – The group formed by C. scaurus, C. porphyropus and C. purpurascens was weakly supported by an a posteriori probability of 56% in the molecular phylogenetic analysis; C. porphyropus and C. purpurascens were linked with an a posteriori probability of 100%. Moser (1960, 1983, 1986) classified these species in Section Laeticolores, subsection Purpurascentes, and Moënne-Loccoz et al (1990–2001) in section Thalliophili, subsection Purpurascentes (C. porphyropus and C. purpurascens) and subsection Thalliophili (C. scaurus). Cortinarius scaurus, C. porphyropus and C. purpurascens appear in a basal position on the tree (Fig. 19) and separate from the main group of subgenus Phlegmacium. Although the clade is weakly supported, characters that justify it include a positive lugol reaction and the change of basidiome coloration when touched or scratched.

ITS sequence variability – ITS sequences in general show little variation at the species level independent of geographical origin. However, in C. glaucopus, C. infractus and C. scaurus, some variation was found among specimens, which might reflect the presence of subspecies or even different (but morphologically very similar) species. Based on macroscopical characters, various subspecies have been recognized in Europe (Moser 1960, Brandrud et al 1990–1998).

Various species with particular morphological, and probably chemical characters, occupied isolated positions in the molecular analysis. These species will have to await the inclusion of a broader spectrum of Cortinarius species in molecular analyses before more meaningful hypotheses about their phylogenetic positions can be derived.

Phylogenetic relationships between sections – Our molecular analyses indicate a close relationship between sections Fulvi (excluding C. mussivus and C. nanceiensis) and Calochroi; they were supported as sister groups with an a posteriori probability of 96%. Representatives of both sections are distinguished by a tendency toward citriform basidiospores, a well-developed gelatinous layer and epicutis, a pileipellis simplex, brightly colored basidiomes and by a marginated, bulbous stipe. Reijnders (1979, 1986) correlated a marginate stipe shape with a pileocarpous development in Phlegmacium species, i.e., a development where the pileus differentiates before the stipe expands. This type of development was considered a derived character by Singer (1986); it might be more efficient in preventing desiccation in early development of basidiomes. In our phylogenetic tree (Fig. 19), sections Fulvi and Calochroi appear separated from the remaining taxa by a relatively large genetic distance. Other possibly derived characters that might be used to confirm this grouping are a well-developed gelatinous layer and epicutis that protect the basidiomes against the rain. Regarding ecology, members of sections Fulvi and Calochroi grow preferentially on basic soil while species of the remaining sections that are included in our study were restricted to acidic soils.

A close relation between sections Glaucopodes and Caerulescentes (C. caerulescens) as reflected in the molecular analysis could be correlated with the micromorphology of the pileipellis (duplex) and gelatinous layer and also with basidiome coloration and stipe shape. However, more species of section Caerulescentes must be included in further analyses to clarify the status of this section. Similarities of stipe shape among species of sections Calochroi, Fulvi, Glaucopodes and Caerulescentes are consistent with the close phylogenetic relationship among these sections that is suggested by our molecular analysis.

Results from molecular and morphological analyses suggest the need to revise the current classification systems for subgenus Phlegmacium in Europe. However, at this time, it would be premature to propose a new classification concept. Other important Phlegmacium species will have to be included in future analyses, and comprehensive morphological, chemical and molecular analyses covering the whole systematic spectrum of Cortinarius species will have to be performed before sound classification concepts can be erected, which very probably no longer will address Phlegmacium as a subgenus but will deal with its present sections in various new phylogenetic subgroups of the fascinating genus Cortinarius.

	ACKNOWLEDGMENTS

 
We thank U. Luhmann, Jena, for providing herbarium material; F. Röger, Troisdorf, and R. Hintzen, Bonn, for samples of fresh Phlegmacia; E. Uhlmann, Tübingen, for critically reading drafts of this manuscript; for financial support to S. G. by the Deutscher Akademischer Austauschdienst (DAAD) through a scholarship.

	FOOTNOTES

 
¹ Corresponding author. E-mail: sigisfredo.garnica{at}uni-tuebingen.de

* In memoriam Meinhard M. Moser, 1924–2002.

Accepted for publication April 24, 2003.

	LITERATURE CITED

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