Mycol Progress (2008) 7:239–247 DOI 10.1007/s11557-008-0565-1

ORIGINAL ARTICLE

Two new species in Cortinarius subgenus Telamonia, Cortinarius brunneifolius and C. leiocastaneus, from Fennoscandia (Basidiomycota, Agaricales) Tuula Niskanen & Kare Liimatainen & Ilkka Kytövuori

Received: 18 February 2008 / Revised: 10 May 2008 / Accepted: 29 May 2008 / Published online: 5 July 2008 # German Mycological Society and Springer 2008

Abstract Two new Cortinarius species, C. brunneifolius and C. leiocastaneus, are described based on molecular and morphological data. Cortinarius brunneifolius looks like a trivial brown Telamonia. It is characterised by the fairly dark brown cap and gills contrasting with the white stem. The major distinctive features are the refracting, colourless granules in the epicutis hyphae, broadly ellipsoid, equally verrucose spores, and fruitbody without bluish tints. Cortinarius leiocastaneus can be fairly easily recognised by the appearance of miniature brown Telamonia species, narrowly amygdaloid spores, and a habitat with Betula. Detailed descriptions of the species are provided as well as comparisons with macro- and microscopically similar existing telamonioid taxa. Phylogenetic relationships within the genus were studied by use of ITS rDNA sequence data. Ten new ITS sequences are published. Cortinarius brunneifolius and C. leiocastaneus are fairly closely related and belong to the subgenus Telamonia sensu stricto. The infrasubgeneric relationships were not well resolved, but the new species seem to be unrelated to the macroscopically similar species and do not clearly belong to any of the existing sections. Taxonomical novelties: Cortinarius brunneifolius Kytöv., Niskanen & Liimat., Cortinarius leiocastaneus Niskanen, Liimat. & Soop. T. Niskanen (*) : K. Liimatainen Department of Biological and Environmental Sciences, Plant Biology, University of Helsinki, P.O. Box 65, 00014 Helsinki, Finland e-mail: [email protected] K. Liimatainen e-mail: [email protected] I. Kytövuori Botanical Museum, University of Helsinki, P.O. Box 7, 00014 Helsinki, Finland

Keywords Taxonomy . ITS . DNA

Introduction Recent phylogenetic studies, based on sequence data from rDNA internal transcribed spacer regions (ITS1 and ITS2) and/or adjacent LSU region, have shown that genus Cortinarius is monophyletic, but many of the traditional infrageneric groups are artificial (Garnica et al. 2005; Høiland and Holst-Jensen 2000; Liu et al. 1997; Peintner et al. 2004; Seidl 2000). Still, many of the species traditionally classified in Telamonia form a monophyletic group, Telamonia sensu stricto (s. str.), which is characterised at the molecular level by one or two indels in the ITS1 region (Garnica et al. 2005; Høiland and Holst-Jensen 2000; Peintner et al. 2004). Several internal groupings of Telamonia have been introduced based on macromorphology (Bidaud et al. 1994; Melot 1990; Moser 1983), but so far no extensive molecular studies have been performed to test these classifications. The centre of diversity of Telamonia s. str. species is in the northern hemisphere. Species recognition in Cortinarius subgenus Telamonia, and also in many other fungi, has until recently relied almost entirely on morphology and ecology. Due to the fairly simple structure of fungi, the morphological characteristics suitable for classification are fewer than in most animal and plant groups. In addition, the fluid transitions among most of these characteristics and the large number of species make the taxonomy of telamonioid species particularly difficult (Peintner et al. 2004). This has led to a variety of different opinions on species delimitation (e.g. Cortinarius section Armillati in Brandrud et al. 1989, 1992; Bidaud et al. 1995). In recent years, the application of molecular markers has provided a powerful tool for species level taxonomy, and

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some studies of Cortinarius including both morphological and molecular data have been published (e.g. Frøslev et al. 2006a, b, 2007; Garnica et al. 2005; Liu et al. 1997; Moser and Peintner 2002a, b; Niskanen et al. 2006b). Only a few of them, however, have dealt with Telamonia species (Ammirati et al. 2007; Kytövuori et al. 2005; Lindström et al. 2008; Matheny and Ammirati 2006; Niskanen et al. 2006a). The difficulty of the group and the lack of molecular studies are the major reasons why most groups in Cortinarius subgenus Telamonia are still very poorly known and even many common species are not yet described (Brandrud et al. 1989; Niskanen et al. 2008). During our extensive study of boreal Telamonia species, we found over 100 species new to northern Europe (Niskanen et al. 2008). Some of them are already described from other areas, but some of them are completely new. In this paper, we describe two of the species as new based on morphological and molecular data. The species concept follows Kytövuori et al. (2005).

Materials and methods A total of 29 specimens from Finland and Sweden were studied (deposited in H, S, TUR, and Soop pers. herb.). The taxonomical descriptions are based on the material collected by the authors including specimens in all stages of age. Macroscopic characteristics were observed from fresh fruitbodies and several collections were used for the descriptions. Representative collections were also photographed in fresh condition. Colour codes were not used; instead photographs of the new species C. brunneifolius and C. leiocastaneus are provided. Microscopic characteristics were observed from dried material mounted in Melzer’s reagent and measurements

were made with a 100× oil immersion lens. Twenty spores, from collections marked with S in specimens examined, were measured from one fruitbody, from the veil or top of stipe. Length and width were measured from the same spore, and the length/width ratios (Q-value) were calculated for individual spores. The extreme measurements were excluded from the final values. The hyphae of the lamellae trama, basidia, and pileipellis were examined as in Kytövuori et al. (2005). Herbarium acronyms follow Holmgren et al. (1990). Three collections of C. brunneifolius and five collections of C. leiocastaneus were sequenced (Table 1, marked with D in the lists of specimens examined). In addition, sequences of the morphologically similar C. inonosmus M.M. Moser, Nespiak & Schwöbel and C. testaceofolius H. Lindstr. & Soop were produced. Total DNA was extracted from a few milligrams of dried material (a piece of gill) using the NucleoSpin Plant kit (Macherey-Nagel). The primers ITS 1F and ITS 4 (Gardes and Bruns 1993; White et al. 1990) were used to amplify the ITS regions of the rDNA. PCR amplifications were performed in a 25-μl reaction mix with about 70 ng of extracted DNA, 1.0 U Phusion High-Fidelity DNA polymerase and 1X HF-buffer (Finnzymes, Finland), 200 μM of each dNTP and 0.4 μM of each primer. The PCR reactions were run on a MBS 0.2G Thermal Cycler (Thermo Hybaid) with the following settings: denaturation for 30 s at 98°C, followed by 35 cycles of: denaturation for 10 s at 98°C, annealing for 30 s at 50°C, and extension for 30 s at 72°C. The PCR products were purified using an ExoSAP-IT purification kit (Amersham Biosciences). Sequencing was performed on both strands using a BigDye Terminator v1.1 Sequencing kit (Applied Biosystems). Reactions were performed in 10 μl with 1 μl of PCR-product, 1.3 μM of primer (ITS 1F or ITS 4), 1 μl 5× sequencing buffer, and 1 μl Terminator Ready Reaction Mix. Reactions were run for 1 min at 96°C,

Table 1 Specimens sequenced in this study (GenBank numbers marked in boldface), and sequences of the studied species in the public databases Species

Voucher

Herb.

Locality

GenBank accession number

C. C. C. C. C. C. C. C. C. C. C.

TN06-146 TN05-054 IK02-018 sp. ectomycorrhiza TN06-150 TN04-763 TN04-854 CO237 CO538 TN02-848 TN02-593

H H H H H H S S H H

Finland, PK, Kitee Finland, EH, Ruovesi Finland, PH, Konnevesi Sweden Finland, PK, Kitee Finland, U, Espoo Finland, U, Porvoo Sweden, Vsm, Arboga Sweden, Hrj, Hede Finland, Ks, Kuusamo Finland, Kn, Suomussalmi

EU259284 EU259282 EU259283 AF476972 EU259289 EU259285 EU259286 EU259287 EU259288 EU693241 EU693242

brunneifolius Kytöv., Niskanen & Liimat. (holotype) brunneifolius brunneifolius brunneifolius leiocastaneus Niskanen, Liimat. & Soop (holotype) leiocastaneus leiocastaneus leiocastaneus leiocastaneus ionosmus M.M. Moser, Nespiak & Schwoebel testaceofolius H. Lindstr. & Soop

For acronyms of biological provinces, see, e.g. Hansen and Knudsen 1992: pp. 24–25

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followed by 30 cycles of: 30 s at 96°C, 15 s at 50°C, and 4 min at 60°C. Unincorporated dye terminators and primers were removed by Sephadex G-50 DNA Grade Fine (Amersham Biosciences) purification and the reactions were analysed by a MegaBace (Amersham Biosciences) automatic sequencer. Sequences were assembled and edited using Sequencher 4.1 (Gene Codes, Ann Arbor). Intragenomic polymorphism was observed as mixed peaks in chromatographic data. Base polymorphisms are marked with ambiguous IUB codes (further information is provided upon request). Intragenomic variation within Cortinarius has previously been reported by Frøslev et al. (2007). In order to understand the closeness of the studied sequences, they were aligned using the ClustalW 1.8 program (Thompson et al. 1994) on the European Bioinformatics Institute server (http://www.ebi.ac.uk/clustalw/ index.html). Differences in and between the species were counted from alignments. The differences between the species were counted as minimum evolutionary events, i.e. multiple base indels are treated as one change. For more information, see point “ITS-regions” in each species. To study the phylogenetic position of C. brunneifolius and C. leiocastaneus, their sequences were compared with our own unpublished sequences and the sequences in the public databases (GenBank: http://www.ncbi.nlm.nih.gov/ and UNITE: http://unite.ut.ee/) using BLAST search as a first step. The ITS sequences showed the highest percentage of similarity with the ITS sequences of other species of subgenus Telamonia s. str. For the preliminary analysis (POY; Gladstein and Wheeler 2001; Wheeler 1996; result not shown) approximately 200 ITS sequences of the species of subgenus Telamonia s. str. were then chosen including our own unpublished and selected sequences retrieved from the public databases. The analysis was run with Cortinarius norrlandicus as an outgroup. Based on these results, 25 Telamonia species representing different clades of subgenus Telamonia and the species which appeared in the tree topology related to C. brunneifolius and C. leiocastaneus were chosen for the final analysis. Only one sequence of C. brunneifolius and C. leiocastaneus was included because the sequences of the former are identical and the sequences of the latter only have intragenomic polymorphism. Three species of subgenus Phlegmacium, C. norrlandicus, C. barbarorum and C. calochrous, were used as outgroup. An alignment of 30 sequences for phylogenetic analysis was produced with Muscle program (Edgar 2004) under default settings and followed by manual adjustments in BioEdit (www.mbio.ncsu.edu/BioEdit/bioedit.html). The original alignment was 653 nucleotides long (including gaps). After the exclusion of areas with ambiguous alignment (positions 195–198, 495–497 and 622–624), 643 positions were used

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for analysis. Alignment is available in TreeBase (http:// www.treebase.org/treebase/index.html). Maximum parsimony (MP) analysis was performed with TNT v.1.0 (Goloboff et al. 2000). The analysis was run using two dual processors of 2.6 GHz. A heuristic search strategy with TBR branch swapping, 10,000 random replicates, and maxtrees of 10,000 was employed. Gaps were treated as a fifth character for dataset. Jackknife (JK) values were counted with 10,000 replicates. Analysis was run with the computer clusters of the CSC, IT Centre for Science, Espoo, Finland.

Phylogenetic results The analysis of ITS sequences resulted in two most parsimonius trees with the length of 857 steps. Figure 1 shows one of the two equally most parsimonious trees as a cladogram. With 10,000 replicates, the shortest trees were found 130 times. Based on the analyses, C. brunneifolius and C. leiocastaneus belong to the subgenus Telamonia s. str. (JK 100%). They clustered together with C. flexipes and C. evernius as their closest relatives but with low (<50%) support values. Only clades Firmiores, Saturnini, and Bovini were supported by jackknife values >80%, and the species pair C. saniosus/C. psammocephalus by JK value 64%, while other relationships were not well resolved.

Taxonomic descriptions Cortinarius brunneifolius Kytöv., Niskanen & Liimat. spec. nov MycoBank no.: MB 511472 Etym: brunneifolius (Latin), brunneus=brown, folium= leaf. The epithet refers to the similarity with C. testaceofolius, but with darker, brown gills (Figs. 2, 3 and 4). Pileus 3.5–6 cm, convexus, dein planoconvexus, obtuse umbonatus, laevis, opacus griseobrunneus, dein radialiter fuscescente-nigrescente maculatus, hygrophanus. Lamellae subconfertae, primo pallide griseobrunneae, dein obscure griseobrunneae. Stipes 6–13×0.6–1.2 cm, clavatus vel cylindraceus, albus, fibrillosus. Velum album, gracile. Caro primo albida, mox pallide brunnea. Odor obsolete raphanoides. Sporae 8–9(−9.5)×5.5–6.3 μm, late ellipsoideae, paulo verrucosae. In silvis coniferis semiaridis et mediocriter humidis, cum Picea abiete. Holotypus. Finland, Pohjois-Karjala, Kitee commune, Hammaskallio, in fairly old, mesic, mossy coniferous forest (Picea, Pinus) with some birch (Betula), 20 Sept 2006,

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Fig. 1 One of the two most parsimonius trees inferred from ITS regions (including 5.8S). Jackknife values >50% are indicated above branches. Bold names indicate the two new species and bold numbers indicate sequences generated in this study. Sequences from type material are marked with (T)

C.armillatus DQ114744 C.brunneus DQ117927(T) C.torvus AY669668 C.laniger AF325592 C.boulderensis DQ499466(T) C.heterosporus AF268894 C.hinnuleus DQ117926

64

C.saniosus DQ102680 C.psammocephalus AY669672

100

C.decipiens AY083180

92

C.armeniacus DQ117925 C.alboviolaceus AF325597

89

C.lucorum AY695794 C.saturninus AY083189

Firmiores

Saturnini

C.evernius AJ236077 C.flexipes AY669683 C.leiocastaneus EU259289(T) C.brunneifolius EU259284(T) C.traganus AF325598 C.malachius AY669681 C.testaceofolius EU693242 C.raphanoides AF389158 C.ionosmus EU693241

83 88 73

C.bovinus DQ139983 C.sordidemaculatus DQ139984(T) C.neofurvolaesus DQ139999(T)

Bovini

C.anisatus DQ117931(T)

100

C.norrlandicus DQ117928(T) C.barbarorum DQ663237 C.calochrous DQ663250

Liimatainen & Niskanen F06-146, EU259284 (H, isotype S and NYBG). Pileus. 3.5–6 cm, hemispherical, then low convex with an obtuse umbo, slightly matt, greyish brown, later with

A

B

10 µm

Fig. 2 Cortinarius brunneifolius, Finland, Pohjois-Karjala, Kitee commune, Hammaskallio, 2006 Liimatainen & Niskanen F06-146a (H, holotype). Photo K. Liimatainen

Fig. 3 Spores of a Cortinarius brunneifolius and b C. leiocastaneus. Drawings T. Niskanen

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Fig. 4 Distribution of Cortinarius brunneifolius in northern Europe according to the material examined

darkening spots, hygrophanous. Lamellae medium spaced (34–42 reaching the stipe), emarginate, moderately broad, pale greyish brown, later fairly dark greyish brown, with white edge. Stipe 6–13×0.6–1.2 cm, clavate or cylindrical, sometimes tapering downwards, white silky-fibrillose. Universal veil white, sparse. Flesh whitish but coming soon pale brownish, e.g. after thumbing or cutting. Mycelium white. Smell faintly raphanoid. Exsiccata with fairly dark, red brown cap, often depressed in the centre, greyish white to brownish stem and often fairly dark brown gills. Spores. 8–9(−9.5) × 5.5–6.3 μm, Q = 1.37–1.60, X¼ 8:2
9:05:6
6:3 μm, XQ¼1:44
1:53 (240 spores, 12 collections, (Fig. 3), ellipsoid to broadly ellipsoid, fairly

finely to moderately, densely and evenly verrucose, somewhat more strongly at the apex, weakly to moderately dextrinoid. Basidia 4-spored, 28–45×8–10 μm (120 basidia, 12 collections), concolorous to brownish, sometimes guttulate. Lamella edge mostly fertile, concolorous, marginal cells clavate, 9–23×5–9 μm (93 cells, 10 collections). Lamella trama hyphae smooth to finely zebra-striped incrusted, pale olivaceous yellowish. Pileipellis duplex: Epicutis light coloured, hyphae of the epicutis ca. 3–10 μm wide, thinwalled, smooth, with numerous, colourless, refracting intracellular granules (ca. 1–3 μm wide), hyphae embedded in a gelatinous substance. Hypodermium present but fairly poorly differentiated, pale brownish, hyphae of the hypodermium 30–55×13–22 μm, thin-walled, smooth or finely

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incrusted. Hyphae of the context 5–23 μm wide, moderately to strongly zebra-striped incrusted. Clamp connections present. Stipitipellis hyphae at the stem top yellowish, somewhat wider than those in the pileipellis, not gelatinose, not incrusted. ITS-regions (including 5.8S region). 504 bases long (a total of three sequences, Table 1). All the sequences are identical and have a clear and strong signal in the chromatographic data for whole ITS-region. The GenBank sequence AF476972 differs by one base, but we have not seen the chromatogram. The difference compared to C. leiocastaneus is at least 18 evolutionary events. Type material. The holotype is abundant including ten fruitbodies and three caps, the isotype (S) includes five fruitbodies, and the isotype (NYBG) includes four fruitbodies and one cap, all in good condition. The holotype includes the photographed fruitbodies. Ecology and distribution. In mesic to dryish coniferous forests, presumably with Picea, often deep in mosses. Known from Finland and Sweden, and considered occasional (Fig. 4). An ectomycorrhizal ITS-sequence from Sweden (GenBank, AF476972) was also identified as this species (Table 1). The sample has been collected from an old coniferous forest dominated by Picea abies and Pinus sylvestris. Comments. Cortinarius brunneifolius looks like a trivial brown Telamonia. Typically, the fairly dark brown cap and gills contrast with the white stem. The major distinctive features are the refracting, colourless granules in the epicutis hyphae, broadly ellipsoid, equally verrucose spores, and fruitbody without bluish tints. The cap is greyish brown when fresh but reddish brown in exsiccata. The species is most reminiscent of C. testaceofolius, which lacks the granules in epicutis hyphae, has a more reddish brown cap and gills, and amygdaloid spores. Cortinarius brunneifolius can also resemble the species in the sect. Bovini M.M. Moser ined., but those usually have darker context, different spores, and sordid brown exsiccata. It can be distinguished from the species of the sect. Firmiores (Fr.) Hennings by darker gills and somewhat darker flesh, darker exsiccata and colourless granules in the epicutis hyphae. Cortinarius brunneifolius can also be confused with C. ionosmus, but the latter has the smell of Viola odorata, longer spores (9–10×5–6) and lacks the colourless granules in the epicutis hyphae. In addition, several nameless Telamonia species occur in the boreal coniferous forests that might be confused with C. brunneifolius, but none of them has the combination of epicutis granules and broadly ellipsoid spores. Cortinarius brunneifolius is a well defined species based on morphology and molecular data. Since we have not found an existing name from the literature, we describe the species as new. Collections examined. Finland: Varsinais-Suomi: Vihti, Lintumäki, 7 Oct 2001, H. Tuovila & I. KytövuoriS (H).

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Etelä-Karjala: Anjalankoski, Kaipiainen, 15 Sept 1994, I. Kytövuori 94-575 (H), loc. cit., 15 Sept 1994, I. Kytövuori 94-578S (H). Etelä-Häme: Ruovesi, S of Musturinlammi, 5 Sept 2005, K. Liimatainen & T. Niskanen 05-054DS (H), Susimäki, 9 Sept 2005, K. Liimatainen & T. Niskanen 05076 (TUR). Etelä-Savo: Juva, Hatsola, 4 Sept 2007, I. Kytövuori & M. Toivonen (H). Pohjois-Häme: Konnevesi, Tuhola, 13 Sept 2002, I. Kytövuori 02-018 DS (H); Töhkerönmäki, 6 Sept 2002, I. Kytövuori (H). Laukaa, Hitonhauta, 8 Sept 2002, I. Kytövuori (H), loc. cit. 10 Sept 2004, I. KytövuoriS (H); Äijälä, 11 Sept 2002, I. KytövuoriS (H, TUR), loc. cit., 10 Sept 2004, I. Kytövuori (H). Saarijärvi, Pyhä-Häkki National Park, 10 Sept 2002, I. Kytövuori S (H). Virrat, Hauhuu, I. Kytövuori, 14 Oct 2001S (H). Pohjois-Karjala: Kitee, Hammaskallio, 20 Sept 2006, K. Liimatainen & T. Niskanen F06-146 DS (holotype, H). Lieksa, Patvinsuo National Park, 21 Sept 1996, S. Huhtinen & mushroom courseS (H). Keski-Pohjanmaa: Pyhäntä, Itämäki, 14 Sept 2005, I. Kytövuori (H). Kainuu: Kuhmo, Elimyssalo, Kivijärvi, 29 Aug 1999, I. Kytövuori (H); Elimyssalo, Riihivaara, 30 Aug 1999, I. KytövuoriS (H). Sweden: Dalarna: Sollerö, Gesunda, 11 Sept 2007, T.B. Borgen (C). Medelpad: Ånge, Borgsjö, 11 Sept 1995, P. & I. Kytövuori 95-1091S (H). Cortinarius leiocastaneus Niskanen, Liimat. & Soop spec. nov MycoBank no.: MB 511473 Etym: leiocastaneus (Greek), leios=glabrous, kastanon= chestnut. The epithet refers to the glabrous, red brown cap (Figs. 3, 5 and 6). Pileus 1.5–4 cm, convexus, dein planoconvexus, obtuse umbonatus, laevis, interdum laeviter translucidus, ochraceo-

Fig. 5 Cortinarius leiocastaneus, Finland, Pohjois-Karjala, Kitee commune, Hammaskallio, 2006 Liimatainen & Niskanen F06-146a (H, holotype). Photo K. Liimatainen

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Fig. 6 Distribution of Cortinarius leiocastaneus in northern Europe according to the material examined

brunneus vel fulvobrunneus, hygrophanus. Lamellae subconfertae, pallide brunneae, dein brunneae. Stipes 3.5–5× 0.3–0.5 cm, cylindraceus vel aliquantum clavatus, albidus fibrillosus, dein pallide brunneus. Velum albidum, sparsum. Caro pallide brunnea, variegata. Odor obsolete raphanoides. Sporae 8–9×4.5–5.2 μm, anguste amygdaliformes, subtiliter verrucosae. In silvis mediocriter humidis, cum Betulis. Holotypus. Finland, Pohjois-Karjala, Kitee commune, Hammaskallio, in fairly young, mesic, mixed forest (Pinus, Picea, Betula), collected under birch (Betula), 20 Sept 2006, Liimatainen & Niskanen F06-150, EU259289 (H, isotype S and NYBG). Synonym. C. erugatus (Weinm.) Fr. ss. Soop.

Illustrations. SOOP 2004, fig. 94 [collection Soop CO237 (Soop pers. herb.)]. Pileus 1.5–4 cm, hemispherical, then low convex, with an obtuse umbo, sometimes narrowly pellucid-striate, glabrous, honey brown to red brown, hygrophanous. Lamellae medium spaced (28–37 reaching the stipe), emarginate, moderately broad, pale brownish, later brown, with whitish edge. Stipe 3.5–5×0.3–0.5 cm, cylindrical to slightly clavate, whitish fibrillose, later more brownish. Universal veil white, sparse. Flesh pale brownish, marbled hygrophanous. Mycelium white. Smell faintly raphanoid. Exsiccata with dark brown cap, greyish to brownish stem and brown to fairly dark brown gills.

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Spores 8–9×4.5–5.2 μm, Q=(1.65–)1.70–1.90(–1.95), X ¼ 8:3
8:74:6
4:9 μm, XQ ¼ 1:73
1:86 (100 spores, 5 collections; Fig. 3), narrowly amygdaloid, fairly finely and somewhat sharply verrucose, fairly weakly to moderately dextrinoid. Basidia 4-spored, 24–35×7–9 μm (40 basidia, 4 collections), concolorous to brownish yellowish, sometimes guttulate. Lamella edge mostly fertile, concolorous, marginal cells not distinguishable from basidioles. Lamella trama hyphae somewhat olivaceous yellowish, smooth to finely incrusted. Pileipellis duplex: Epicutis fairly thin, hyphae of the epicutis ca. 3–9 μm wide, smooth to very finely incrusted, with some colourless, refracting granules (ca. 1–3 μm wide). Hypodermium present, very pale brownish, hyphae of the hypodermium 35–50×13– 30 μm, very finely incrusted. Hyphae of the context ca. 6– 25 μm wide, moderately to strongly zebra-striped incrusted. Clamp connections present. Stipitipellis hyphae at the stem top smooth to very finely incrusted, ca. 3–9 μm wide. ITS-regions (including 5.8S region). 500 bases long (a total of five sequences; Table 1). All the sequences have a clear and strong signal in chromatographic data for whole ITS-region. An intragenomic polymorphism occurred altogether 8 times in 4 different sites in 4 different specimens. More specimens and more variable DNA-regions would be needed to study this genetic variation more closely. We did not find sequences of this species from the public databases. Difference to C. brunneifolius is at least 18 evolutionary events. Type material. The holotype is abundant including six fruitbodies and two caps, the isotype (S) includes four fruitbodies, and the isotype (NYBG) includes three fruitbodies, all in good condition. The holotype includes the photographed fruitbodies. Ecology and distribution. In hemiboreal to boreal, mesic, mixed forests usually with Betula, in mosses. Often solitarily in small groups. Known from Finland and Sweden (Fig. 6) and considered rare, but possibly overlooked. Fruiting in late summer to autumn. Comments. Cortinarius leiocastaneus can be fairly easily recognized by the appearance of miniature C. ionosmus or C. brunneifolius, narrowly amygdaloid spores, and a habitat with Betula. Some unpublished species in the sect. Bicolores can resemble C. leiocastaneus, but the former usually presents bluish tints in the fruitbody, paler exsiccata, and grows with coniferous trees. Cortinarius leiocastaneus differs from the species of the sect. Hydrocybe and Incrustati by not having strongly incrustated epicutis or hypoderm. Soop (2004) used the name C. erugatus (Weinm.) Fr. for this species. The Weinman’s protologue is too short and vague to be conclusive, and in Fries (1838) C. erugatus is said to have a lilac stem top, a character that we have never observed in C. leiocastaneus. As we have not found any

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reliable name for our species in the literature we publish it here as new. Since the species is small and probably rare, it is likely that it has not been previously reported. Collections examined. Finland: Uusimaa: Espoo, Luukkaa outdoor recreation area, N of Hauklampi, 9 Sept 2004, K. Liimatainen & T. Niskanen F04-763 D (H), loc. cit. 04-765 (TUR), loc. cit. 04-771 (H). Porvoo, Venjärvi, 17 Sept 2004, K. Liimatainen & T. Niskanen 04-854 D (H). PohjoisKarjala: Kitee, Hammaskallio, 20 Sept 2006, K. Liimatainen & T. Niskanen F06-150 D (H, holotype). Sweden: Västmanland: Arboga, Ramstigsberget, 25 Aug 1987, K. Soop CO237 D (Soop pers. herb.). Härjedalen: Hede, golf course, 12 Aug 1992, K. Soop CO538 D (Soop pers. herb.), loc. cit. 17 Aug 1988, K. Soop CO331 (Soop pers. herb.).

Discussion Based on morphology and molecular data Cortinarius brunneifolius and C. leiocastaneus are well defined species. The observed intraspecific variation in the studied species was very low and correlated well with the results by, e.g., Frøslev et al. (2007) and Kytövuori et al. (2005). Based on our phylogenetic analysis, C. brunneifolius and C. leiocastaneus belong to the subgenus Telamonia s. str. The infrasubgeneric relationships were not well resolved except for the clades Firmiores, Saturnini and Bovini. Based on the preliminary results it seems, however, that C. brunneifolius and C. leiocastaneus could be fairly closely related. Furthermore, they seem to be unrelated to the macroscopically similar species and do not clearly belong to any of the existing sections. Further studies with more variable molecular regions and higher number of species would be needed for gaining more reliable phylogeny of Telamonia species. Acknowledgements We thank Heino Vänskä for revision of the Latin descriptions and referees for valuable comments. This work was supported by the Ministry of Environment, Finland (YM195/5512/ 2002-5 & YM196/5512/2002-5).

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