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Institute of Microbiology, Leopold Franzens-University Innsbruck, Technikerstr. 25, 6020 Innsbruck, Austria
Egon Horak
Geobotanical Institute, ETHZ, Zollikerstr. 107, Zürich CH-8008, Switzerland
Meinhard M. Moser
Institute of Microbiology, Leopold Franzens-University Innsbruck, Technikerstr. 25, 6020 Innsbruck, Austria
Rytas Vilgalys
Department of Biology, Duke University, Durham, North Carolina 27708
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ABSTRACT |
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Phylogenetic relationships of Rozites, Cuphocybe, and Rapacea were assessed using molecular phylogenetic approaches. These three genera are placed in Cortinariaceae and have been regarded as closely related to Cortinarius. Rozites includes more than 20 species, which are characterized by having both a membranaceous partial veil in the form of a persistent annulus and a membranaceous universal veil. Cuphocye (4 species) lacks an annulus or cortina, but has pigmented veil fibrils or scales. The monotypic genus Rapacea accommodates a distinct taxon with pale, nearly smooth and thick-walled basidiospores. We analyzed 56 sequences of the internal transcribed spacer region (ITS1, ITS2, and the intervening 5.8S rRNA gene) for nine species of Rozites, three species of Cuphocybe, 28 species of Cortinarius, Rapacea mariae and Protoglossum luteum. Two species of Hebeloma were used as outgroup. Large subunit (LSU) rDNA sequences from selected taxa were also analyzed. The results clearly demonstrate that Rozites species are nested within the clade/Cortinarius, and that Rozites is polyphyletic, suggesting that membranaceous veils have evolved several times in the genus Cortinarius. Also Rapacea and Cuphocybe are nested within Cortinarius, making the latter genus paraphyletic. Based on phylogenetic studies, Rozites, Cuphocybe and Rapacea are artificial genera and do not reflect natural relationships.
Key words: Agaricales, amyloid reaction, Cortinarius, Dermocybe, Protoglossum
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Halling and Ovrebo 1987, Bougher et al 1994).
Separation of Cortinarius Fr. from Rozites is difficult, especially in the Southern Hemisphere, and many species of Cortinarius with an annulus or a volva have been described from this region. Taxonomic uncertainty involving these genera and problems in species delimitation are reflected in the transfer of taxa between Cortinarius and Rozites. For example, Rozites collariatus was initially placed in Cortinarius subgenus Myxacium (Fr.) Trog because it possesses an inconspicuous universal veil (Moser and Horak 1975). This species was regarded as transitional taxon between Rozites and Cortinarius, making the morphological delimitation of Rozites more difficult (Moser and Horak 1975). In contrast, Rozites australiensis Cleland & Cheel was recombined with Cortinarius on the basis of its arachnoid, but not membranaceous, partial veil (Horak 1981).
The boundaries between Rozites and other members of the Cortinariaceae are also unclear. Differences between Cuphocybe R. Heim and Rozites are indistinct and restricted to veil characters. The presence or absence of an annulus is the main morphological character separating these genera, and Cuphocye is characterized further by its pigmented veil fibrils or scales and by the lack of a cortina (Heim 1951, Horak 1973, Singer 1986, Soop 1998). To date, six species of Cuphocybe have been described from New Zealand or China, and most are considered to be ectomycorrhizal symbionts of Nothofagus or Quercus (Heim 1951, Horak 1973, 1980, Zang 1987, Soop 1998).
The separation of Descolea Singer and Rozites on the basis of macroscopic characters also presents difficulties. However, these two genera are distinct microscopically and Singer (1986) excluded the possibility of a close relationship between Descolea and Rozites on the basis of these characters. Comparison of ITS sequences demonstrates that Descolea is a monophyletic genus and not closely related to Rozites (Peintner et al 2001). We conclude, therefore, that macroscopically similar basidiomes have arisen independently in these lineages.
Recently, the monotypic genus Rapacea E. Horak was erected to accommodate R. mariae, a species closely resembling members of Cortinarius stirps Cystidiorapaceus that occurs in New Zealand, Tasmania, and Papua New Guinea (Horak 1999). Rapacea mariae is characterized by thick-walled basidiospores that appear smooth when observed by light microscopy but which are minutely asperulate when examined by scanning electron microscopy. Furthermore, the spore print of R. mariae is olive and not rusty brown in color as is typical for the majority of species of Cortinarius. Rapacea was regarded as a rather isolated genus closely related to Cortinariaceae (Horak 1999).
The aim of the present study was to ascertain the phylogenetic relationships of Rozites, Cortinarius, Cuphocybe, and Rapacea based on the analysis of rDNA sequence data. Earlier results have demonstrated that Rozites caperatus is nested within Cortinarius (Høiland and Holst-Jensen 2000) and that Cuphocybe melliolens is closely related to Cortinarius (Peintner et al 2001). In this study, we expanded our sampling of Cuphocybe and Rozites to investigate whether these genera are monophyletic, and to explore their relationships to Cortinarius and other Cortinariaceae. We also examined the amyloid reactions of tissues of a number of taxa included in this study to assess the utility of this character in the delimitation of Rozites.
Furthermore, we wanted to test the current hypothesis on the origin and biogeography of Rozites within a phylogenetic framework. This hypothesis (based on distributional data) (Horak and Taylor 1981, Bougher et al 1994) assumes the origin of Rozites within the Southern Hemisphere, spanning the geographical range of ectomycorrhizal plant taxa of the Cretaceous fagalean complex. Examining the biogeography of Rozites may result in new insights concerning the origin, mechanisms of dispersal, and biogeography of ectomycorrhizal basidiomycetes in general.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. Samples of dried basidiomes were homogenized with a plastic pestle in a microcentrifuge tube (1.5 mL) containing 300–500 µL 2x CTAB lysis buffer (2% w/v CTAB, 100 mM Tris pH 8, 20 mM EDTA, 1.4 M NaCl). After soaking for several hours at room temperature, the tubes were incubated in 70 C for 30 min. An equal volume of chloroform was added, and the contents of the tubes were vortexed and then centrifuged at 10 000 g for 15 min. The aqueous phase was transferred to a new tube, purified either by ethanol/isopropanol precipitation or Geneclean no. III (Bio 101, La Jolla, California) and resuspended in 55 µL ddH2O. The DNA extracts were diluted to 0.1–1.0 ng/µL and used as templates for the polymerase chain reaction (PCR).
Amplifications employing PCR were conducted using standard protocols in a final volume of 25 µL employing a Perkin Elmer 480 thermocycler (Applied Biosystems, Foster City, California). The primers ITS1 and ITS4 (White et al 1990) were used for amplification and sequencing of ITS region. Amplification and sequencing primers for the LSU included LROR, LR3R, LR5, LR16, and LR7 (Moncalvo et al 2000).
PCR products were quantified using gel electrophoresis on a 0.8% agarose gel stained with ethidium bromide, and purified by microfiltration using Ultrafree-MC centrifugal columns (Millipore, Bedford, Massachusetts). Sequencing was performed using fluorescent dye terminator chemistries following the manufacturer's instructions (Applied Biosystems) employing automated sequencers (ABI 377, ABI 3700, Applied Biosystems, Foster City, California).
Analytical methods –
Sequence chromatograms were edited and contigs assembled using Sequencher 3.0 (GeneCodes, Ann Arbor, Michigan). Sequences are deposited in GenBank (Accession Numbers AY033096–AY033239). Nucleotide sequences were aligned manually in the data editor of PAUP* 4.0d64 (Swofford 1999). The data matrices were deposited in TreeBASE as SN1004–2774 and SN1004–2776.
Maximum parsimony analyses (MP) (heuristic searches) were performed in PAUP* using the following settings: MULPARS = on, steepest descent not in effect, MAXTREES 25000, transitions twice the weight of transversions, gaps treated as missing data. The most parsimonious trees (MPT) were searched with tree-bisection-reconnection (TBR) branch swapping. Starting trees were obtained by random sequence addition; 100 heuristic searches were performed and the shortest trees over all replicates were kept and assumed to be the most parsimonious reconstruction. Relative robustness for individual branches was estimated by bootstrap analysis using 100 replicates (heuristic searches; 10 random addition sequences; TBR; MAXTREES set to 25000).
To test monophyly of Cuphocybe and Rozites, constrained and unconstrained topologies were compared in PAUP* using the Templeton test (Templeton 1983) and the Kishino-Hasegawa test (Kishino and Hasegawa 1989). The program Modeltest version 3.06 (Posada and Crandall 1998) was used to test the model of DNA substitution. Maximum likelihood (ML) analysis was carried out using one MPT as starting tree for TBR branch swapping and employing the likelihood settings from the best-fit model selected by Modeltest. Bootstrap support for branches obtained in ML searches was estimated by 2000 replicates using the "fast" stepwise addition option in PAUP*.
The LSU data as well as the combined data set consisting of 14 ITS and LSU sequences were analyzed as mentioned above. Gapped regions of the alignments were excluded from analyses. Combinability of the ITS and LSU data sets was assessed using the partition homogeneity test (PHT) in PAUP* with 100 replicates.
Amyloid reaction of hyphae –
Tissues of all our Rozites collections were checked microscopically and macroscopically with Melzers reagent (Clemençon 1971, Moser 1983) for evidence of an amyloid reaction.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Phylogenetic analysis of ITS sequences – Parsimony analysis of the 58 ITS sequences yielded 490 MPT (L = 594 steps, CI = 0.452, RI = 0.708, RC = 0.319). The best fitting ML model selected by Modeltest is the Hasegawa-Kishino-Yano model with gamma distributed site-to-site rate variation (HKY+G). The estimated transition/transversion ratio is 2.2681, the gamma-shape parameter is 0.4014, the base frequencies are A = 0.2399, C = 0.1884, G = 0.1909, T = 0.3808, and the proportion of invariable sites is zero. There is no conflict between the tree resulting from likelihood analysis (-Ln = 3747.51699) (Fig. 1) and the MPT (not shown).
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The nine species of Rozites included in this study, three species of Cuphocybe, as well as Rapacea mariae are nested within the genus Cortinarius (Fig. 1). Moreover, the genus Rozites is polyphyletic because the members of this genus belong to both subclades of clade/Cortinarius (subclade/telamonia and subclade/cortinarius). Within the subclade/telamonia, Rozites violacea, R. ochraceoazurea, and R. sarmienti form a clade that is closely related to an undescribed species of Cortinarius collected in South America by Terry Henkel (collections TH 6417 and TH 7472). The six other species of Rozites are nested in the subclade/cortinarius: R. caperatus groups with R. meleagris, R. castanella groups with Cortinarius taylorianus, and R. pallida groups with R. fusipes. Different collections of R. collariatus form a well-supported group (bootstrap value 93%) within the subclade/cortinarius without known relationships.
Strict monophyly of the nine Rozites species was rejected by the Kishino-Hasegawa test and by the Templeton test (P < 0.0001) (Table II). Monophyly of the six Rozites species nested within the subclade/cortinarius was also rejected (P < 0.0209). However, by relaxing the constraints through the inclusion of C. taylorianus, a monophyletic group that includes some species of Rozites within the/cortinarius subclade could no longer be rejected (Table II).
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Rapacea mariae is also positioned within Cortinarius and is a sistergroup to an undescribed Cortinarius species from New Zealand (NZ 8682).
Phylogenetic analysis of LSU sequences –
Parsimony analysis of LSU sequences was carried out for a restricted subset of 14 taxa, resulting in 30 MP trees (L = 176 steps, CI = 0.796, RI = 0.577, RC = 0.459) (Fig. 2). Due to the small number of parsimony-informative characters, the topology of these trees is not resolved, and only the branches supporting the subclade/telamonia and the subclade including C. violaceus and C. hercynicus are supported statistically (bootstrap values 
70%). Thus, LSU sequences provide limited resolution for inferring relationships within the clade/Cortinarius.
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Amyloid tissue reaction of Rozites species – We tested tissues of a number of species of Rozites and Cortinarius for amyloid reactions. Rozites castanella, R. collariatus, R. fusipes, R. ochraceoazurea, R. pallida, R. violacea, R. sarmienti, C. paradoxus, and C. taylorianus possess non-amyloid tissues. The amyloid reaction was found only in R. meleagris and R. caperatus.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The results of this study also allow us to reject the monophyly of the six species of Rozites nested within the subclade/cortinarius (Table II). However, a smaller monophyletic group of species around R. caperatus (including C. taylorianus) could not be rejected. The hyphae of R. caperatus have been studied in detail (Clemençon 1971) and are described as amyloid incrusted. The amyloid reaction can be observed microscopically and macroscopically in both fresh and dried material. Hyphae with amyloid incrustation have also been described for R. colombiana Halling & Ovrebo, a species with violaceous lamellae that occurs in Quercus forests in Colombia (Halling and Ovrebo 1987) and Costa Rica (Halling pers com), and which closely resembles R. caperatus. With the exception of R. caperatus and R. meleagris, all of the taxa included in the present study possess non-amyloid tissues. The taxa with amyloid tissue reactions, R. caperatus and R. meleagris from New Zealand, have a common ancestor (Fig. 1). Morphologically, R. meleagris can be differentiated from R. caperatus by its deep lilac lamellae, glutinous pileus and persistent veil remnants that give the surface of the pileus a characteristic speckled appearance. Thus, we conclude that while amyloid reaction is a valuable character that defines a restricted number of species (R. caperatus, R. meleagris, and R. colombiana), it does not delimit the group of species with rozitoid morphology within the subclade/cortinarius.
The monophyly of Rozites is also not supported by morphological data since the combination of characters that defines Rozites, namely fleshy basidiomes with an annulus and a membranaceous universal veil (Moser and Horak 1975), are also known from Cortinarius spp.. For example, annulate taxa occur in Cortinarius subgenus Phlegmacium (Fr.) Fr., the taxon into which Cortinarius australiensis was placed following its transfer from Rozites (Horak 1981, Bougher and Syme 1998). Other species with membranaceous annuli have been described in Cortinarius subgenus Phlegmacium (Bougher and Hilton 1989), thus further eliminating the distinction between cortinate and annulate taxa in the clade/Cortinarius.
The development of annulus-like structures in the Cortinarii also probably occurred several times. For example, Cortinarius subgenus Paramyxacium M. Moser & E. Horak (type species C. paradoxus) was erected to accommodate species with membranaceous annuli, dry or glutinous stipes, comparatively fragile basidiomes and membranaceous (rather than fleshy), strongly hygrophanous pilei (e.g., C. taylorianus) (Moser and Horak 1975, Horak and Wood 1990). In contrast, annulate Cortinarius spp. of subgenus Myxacium stirps Archeri are characterized by annulus-like partial veils combined with a viscid universal veil and non-hygrophanous, more fleshy pilei. However, transitions between a typical cobweb-like cortina and an annulus can be observed within taxa of stirps Archeri, even within one population (Moser and Horak 1975), and the glutinous veil of C. taylorianus often forms a persistent, membranaceous ring in dry weather (Horak and Wood 1990).
In summary, it is evident that the genus Rozites can no longer be convincingly delimited from Cortinarius on the basis of morphological characters. The description of new species combined with an awareness of the morphological variability within the Cortinariaceae has resulted in a gradual blurring of generic characters used to define Cortinarius and Rozites. All transitional stages from cobweb-like to glutinous or to annulus-like partial veils have been observed in fleshy Cortinarius species, making the clear distinction of Cortinarius and Rozites impossible. We conclude that Rozites can no longer be regarded as a distinct genus.
Biogeography of Rozites – Our results demonstrate multiple origins of Rozites and permit us to reject the hypothesis of a common origin of all of the members of this genus in the Southern Hemisphere. One group of Rozites evolved in South America from a common ancestor within the subclade/telamonia, whereas the other six species of Rozites have origins within the subclade/cortinarius. Interestingly, all Rozites species from New Zealand are nested within the subclade/cortinarius. The only species occurring in the Northern Hemisphere, R. caperatus, is derived from the common ancestor of R. meleagris, a species from New Zealand. Weak support for basal relationships within the subclade/cortinarius prevents us from inferring a geographical pattern for the evolution of these taxa. Further sampling will be necessary to assess whether or not Southern Hemisphere species of Cortinarius form one or more monophyletic groups.
Phylogeny of Cuphocybe and Rapacea – Cuphocybe is also nested within Cortinarius, and Cuphocybe melliolens is closely related to Protoglossum luteum and Cortinarius corrugatus. From a phylogenetic point of view, there are two possible scenarios for the development of the veil characters distinguishing this genus: i) Cuphocybe is derived from Rozites-like precursors through loss of the membranaceous annulus (velum partiale); ii) Cuphocybe is derived from Cortinarius-like precursors by developing a velum universale, which breaks up into squamules. The fact that Cuphocybe melliolens and Cortinarius corrugatus are closely related favors the second hypothesis.
Phylogenetic analyses also show that Rapacea mariae belongs to Cortinarius and that it is closely related to an undescribed species of Cortinarius from New Zealand (#NZ 8682). These taxa share only a few distinctive macro-morphological features, but both possess basidiospores that are weakly ornamented and similar in shape and size.
Morphological plasticity of Cortinarius –
The genus Cortinarius includes species that exhibit a wide range of morphological forms. A number of sequestrate taxa have been shown to have multiple origins within Cortinarius (Peintner et al 2001), and it is now evident that cortinarioid ancestors have given rise to taxa with morphologically distinct basidiomatal types. These are currently circumscribed as Cortinarius, Rozites, Cuphocybe, Rapacea and as the sequestrate genera Protoglossum Massee, Quadrispora Bougher & Castellano, Thaxterogaster Singer, and Hymenogaster Vittad. p.p. (Peintner et al 2002). One striking example of this morphological plasticity is R. ochraceoazurea, which because of variability in the form of its basidiome, was described as a species of Thaxterogaster and then recombined as a species of Rozites with synonyms classified in Cortinarius subgenus Paramyxacium (Moser and Horak 1975, Horak 1979).
This study demonstrates that the genera Rozites, Cuphocybe, and Rapacea cannot be regarded as independent, monophyletic lineages. All are nested within the genus Cortinarius and are either polyphyletic and/or closely related to species of Cortinarius. Retaining these genera would result in the reduction of these taxa to small, monophyletic entities around the type species. Moreover, the genus Cortinarius would be paraphyletic. We therefore recommend synonymizing the genera Rozites, Cuphocybe and Rapacea with Cortinarius.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication January 25, 2002.
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LITERATURE CITED |
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