Mycol Progress (2011) 10:101–105 DOI 10.1007/s11557-010-0680-7

ORIGINAL ARTICLE

Cortinarius badiolaevis, a new conifer-associated, darkening species in the subgenus Telamonia (Basidiomycota, Agaricales) Tuula Niskanen & Kare Liimatainen & Rafael Mahiques & Josep Ballarà & Ilkka Kytövuori

Received: 28 January 2010 / Revised: 2 May 2010 / Accepted: 5 May 2010 / Published online: 28 May 2010 # German Mycological Society and Springer 2010

Abstract A new species, Cortinarius badiolaevis Niskanen, Liimat., Mahiques, Ballarà, Kytöv. in subgenus Telamonia, is described based on morphological and molecular data. Typical for the species is a dark brown cap, pale flesh, indistinct universal veil, small amygdaloid to ellipsoid spores [7.0–8.5×4.5–5.0(−5.5) µm], and dark exsiccata. It occurs in mesic to dryish coniferous forests, and grows in calcareous soil presumably with Pinus and Picea. So far, it is only known from Spain and Sweden, and considered rare. Based on the phylogenetic analyses of the ITS regions, it does not belong to any of the known sections, nor does it have any very closely related sister species.

Taxonomical novelties Cortinarius badiolaevis Niskanen, Liimat., Mahiques, Ballarà, Kytöv. T. Niskanen (*) : K. Liimatainen Department of Biosciences, Plant Biology, University of Helsinki, P.O. Box 65, 00014 Helsinki, Finland e-mail: [email protected] K. Liimatainen e-mail: [email protected] R. Mahiques Dr. Climent, 26, 46837 Quatretonda, Spain e-mail: [email protected] J. Ballarà Tossalet de les Forques, 44, 08600 Berga, Spain e-mail: [email protected] I. Kytövuori Botanical Museum, University of Helsinki, P.O. Box 7, 00014 Helsinki, Finland

Keywords Taxonomy . ITS . DNA

Introduction The subgenus Telamonia, comprising the greatest number of species, is the most poorly known of the subgenera of Cortinarius. Based on the recent molecular studies using mainly the molecular markers ITS1 and ITS2, the previous delimitations of the subgenus have proven to be partly artificial (Garnica et al. 2005; Høiland and Holst-Jensen 2000; Liu et al. 1997; Peintner et al. 2004; Seidl 2000). The majority of the telamonioid species, however, seem to form a monophyletic group Telamonia sensu stricto (s. str.) (Garnica et al. 2005; Høiland and Holst-Jensen 2000; Peintner et al. 2004). The large number of species and similarity in morphological features of those species make the taxonomy of telamonioid species particularly difficult. Therefore, many species have been described more than once while other sometimes common species are not yet described (Brandrud et al. 1989; Niskanen et al. 2008a). Recently, the use of molecular data has facilitated taxonomic work. Several studies on Telamonia species combining morphological and molecular data have been published by, e.g., Ammirati et al. (2007), Kytövuori et al. 2005, Lindström et al. (2008), Matheny and Ammirati (2006), Niskanen et al. (2006, 2008b, 2009), and SuárezSantiago et al. (2009). The molecular markers used have been the rDNA ITS1 and ITS2 regions, which have also proved to be suitable species-level markers in other genera of Agaricales (e.g. Larsson and Jacobsson 2004; Vellinga et al. 2003).

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The aim of this paper is to describe a new Telamonia species based on morphological and molecular data. The species concept follows Kytövuori et al. (2005) and Niskanen et al. (2009).

Materials and methods A total of four specimens from Sweden and Spain were studied (deposited in H, S, and Mahiques, personal herbarium). The taxonomical description is based on material collected by the authors including specimens in all stages of development. Macroscopic characteristics were observed from fresh basidiomata and all the collections were used for the description. Collections were also photographed in fresh condition. Colour codes were not used to describe colours; however, a colour photograph of C. badiolaevis is provided. Microscopic characteristics were observed from dried material mounted in Melzer´s reagent (MLZ) or 3% KOH and measurements were made with an object micrometer using a ×100 oil immersion lens. Twenty spores were measured from one fruitbody from each collection, 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 hyphae of the lamellar trama were examined from the pieces of lamella as well as basidia, which were measured from the same lamellar pieces. In addition, the pileipellis structure was studied from both radial freehand sections and scalps from midway to the pileus centre. The measurements were made from scalp preparations. Herbarium acronyms follow Holmgren et al. (1990). All four collections of C. badiolaevis were sequenced. In addition, a sequence from a collection belonging to the morphologically similar C. uraceus Fr. was produced (HM017844). Total DNA was extracted from a few milligrams of dried material (a piece of lamella) 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. The same primer pairs were used in direct sequencing. PCR amplification and sequencing followed Niskanen et al. (2009). Sequences were assembled and edited using Sequencher 4.1 (Gene Codes, Ann Arbor). To study the phylogenetic position of C. badiolaevis, the 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 (TNT v.1.0; Goloboff et al. 2000; result not shown) approximately 200

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ITS sequences of the species of subgenus Telamonia s. str. were then chosen, with Cortinarius norrlandicus as an outgroup. From the results of this preliminary analysis, 30 Telamonia species were chosen for the final analysis with the following criteria: represent various clades of subgenus Telamonia, have similar morphology to and appear related to C. badiolaevis by tree topology. Three species of the subgenus Phlegmacium, C. norrlandicus, C. barbarorum and C. calochrous, were used as outgroup. An alignment of 33 sequences for phylogenetic analysis was produced using MUSCLE (Edgar 2004) under default settings followed by manual adjustments using BioEdit (www.mbio.ncsu.edu/ BioEdit/bioedit.html). The original alignment was 673 nucleotides long (including gaps). After the exclusion of areas with ambiguous alignment (positions 247–251, 257– 261, 604–607 and 629–635), 652 positions were used for the analysis. Alignment is available in TreeBase (http:// www.treebase.org/treebase/index.html). Maximum parsimony (MP) analysis was performed with TNT v.1.0. A heuristic search strategy with TBR branch swapping, 10,000 random replicates, and maxtrees of 10,000 was employed. Gaps were treated as missing data for the dataset. Jackknife (JK) values were counted with 10,000 replicates. Bayesian inference (BI) was done with the program Mr. Bayes v.3.1.1 (Huelsenbeck and Ronquist 2003). The whole dataset was analysed with the GTR-model including a gamma shape parameter and estimating the proportion of invariable sites. Two independent runs with 4 chains in each were carried out with 1,000,000 generations and sampling at every 100th generation. All trees sampled before stationarity were discarded using a 25% safety margin (burn-in of 2,500 trees (250,000 generations)). The sampled trees from both runs were combined in a 50% majority rule consensus phylogram with posterior probabilities (PP). All analyses were run with the computer clusters of the CSC, IT Centre for Science, Espoo, Finland.

Results of phylogenetic analyses The 50% majority rule phylogram resulting from the BI analysis of the whole data is shown in Fig. 1 with posterior probabilities indicated above the branches. Trees from MP analysis are not shown. The topology of MP trees corresponds with the BI tree in parts marked by the JK supports below the branches. Based on the analyses, C. badiolaevis belongs in the subgenus Telamonia s. str. (100 PP, 99 JK). In the MP analysis, it sometimes clustered together with C. malachius but with a low (<50%) support value. Only clades Saturnini (100 PP, 77 JK), Bovini p.p. (100 PP, 86 JK) and Firmiores (100 PP, 64 JK) were supported in both analyses.

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Fig. 1 The Bayesian 50% majority-rule consensus tree inferred from ITS regions. PP >50 are indicated above branches and JK supports are marked below the branches. Bold name indicates the new species and

bold numbers indicate sequences generated in this study. (T ) Sequences from type material

Taxonomic description

hygrophanus. Lamellae primo ochraceobrunneae, dein brunneae. Stipes 3.5–7.5×0.6–0.9 cm, aliquantum clavatus, albus, fibrillosus. Velum album, sparsum. Caro stipitis albidobrunnea vel pallide brunnea. Odor nullus vel obsolete raphanoides. Sporae 7.0–8.5×4.5–5.0(−5.5) µm, amygdaliformes vel ellipsoideae, subtiliter vel modice verrucosae. In silvis coniferis, fortasse cum Picea abiete et Pino sylvestri in solo calcareo.

Cortinarius badiolaevis Niskanen, Liimat., Mahiques, Ballarà, Kytöv. spec. nov. (Figs. 2 and 3) MycoBank no.: MB 518041 Etymology badius (Latin) = reddish brown, chocolate brown, laevis (Latin) = smooth. The epithet refers to the smooth, brown to dark reddish brown pileus. Formal description Pileus 2–5 cm, convexus, dein planoconvexus, obtuse umbonatus, brunneus vel rubroumbrinus,

Holotypus Sweden, Upland, Närke commune, Lillkyrka parish, Ekebergsbrottet, mesic coniferous forest (Picea, Pinus) on calcareous ground, 26 Sept 2004, Kytövuori, Liimatainen & Niskanen F04-960, HM017846 (H, isotype NY).

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lamellar trama to olivaceous brownish in MLZ and KOH. Lamellar trama hyphae smooth to finely encrusted in MLZ and KOH. Pileipellis duplex: epicutis with hyphae of approx. 3–8 µm wide, thin-walled, smooth, some with pale olivaceous brownish granulose content in MLZ and KOH. Hypodermium present, elements of the hypodermium 30– 55×13–18 µm, thin-walled, smooth. Hyphae in the transition to trama 4–15 µm wide, in MLZ moderately to strongly zebra-striped or spot-like encrusted, pale brownish, in KOH hyaline and encrustations only seen in some hyphae. Clamp connections present.

Fig. 2 Cortinarius badiolaevis, Sweden, Upland. Närke commune, Ekebergsbrottet, 2004 Kytövuori, Liimatainen & Niskanen F04-960 (H, holotype). Photo K. Liimatainen

Description Pileus 2–5 cm, hemispherical, then low convex to almost plane, with an obtuse umbo, waxy-glossy when moist, smooth, margin whitish fibrillose, brown to dark reddish brown, hygrophanous, often drying with a zone around the umbo, then in radial streaks. Lamellae medium spaced (35–42 reaching the stipe), emarginate, moderately broad, yellowish brown, later brown. Stipe 3.5–7.5×0.6– 0.9 cm, slightly clavate, whitish silky-fibrillose. Universal veil white, very sparse. Flesh in stipe whitish brown to pale brownish, in pileus darker. Basal mycelium white. Smell indistinct or very faintly raphanoid. Exsiccata with blackish brown to blackish pileus, brown lamellae, greyish to greyish brown stipe and white basal mycelium. Chemical reactions In the context of pileus guayac slowly and weakly bluish, fenolaniline vinaceous red. Spores 7.0–8.5 × 4.5–5.0(−5.5) µm, av. = 7.3–8.1 × 4.6– 5.1 µm, Q=1.40–1.70, Q(av.)=1.51–1.62 (measurements in MLZ, 80 spores, 4 collections, Fig. 3), amygdaloid to ellipsoid, finely to moderately verrucose, slightly stronger ornamented at the apex, moderately to strongly dextrinoid, somewhat dark in KOH, in MLZ somewhat thick-walled. Basidia 4-spored, 25–35×7–9(−10) µm, concolorous with

Fig. 3 Spores of Cortinarius badiolaevis (Kytövuori, Liimatainen & Niskanen F04-960, H, holotype). Drawing T. Niskanen

ITS-regions (including 5.8 S region) All the sequences are identical (a total of four sequences). We did not find sequences of this species in the public databases. The closest species is C. malachius and it differs by more than 20 evolutionary events. Ecology and distribution In mesic to dryish coniferous forests, presumably with Pinus and Picea, in calcareous soil. Known from Spain and Sweden, and considered rare. Differential diagnosis Cortinarius badiolaevis is recognised by the combination of dark brown pileus, pale flesh, indistinct veil, small amygdaloid to ellipsoid spores, dark exsiccata, and habitat with coniferous trees on calcareous soil. It most closely resembles the species in sect. Bovini M. M. Moser ined., but those usually have a darker context, especially in the base of the stipe. Most species of the section also have distinctly larger spores. The smaller spored C. sordidemaculatus Rob. Henry (syn. C. furvolaesus H. Lindstr.) and C. neofurvolaesus Kytöv., Niskanen, Liimatainen & H. Lindstr. differ by having a distinct veil and spores over 5 µm wide. It can be distinguished from the species of sect. Uracei Kühner & Romagn. ex Melot (Melot 1990) by the basidiomata lacking metallic tints, and from the species of sect. Brunnei Kühner & Romagn. ex Melot ss. Niskanen et al. (2009) by the different spores. The spores of the Brunnei species are more strongly verrucose and/or have a different shape. In addition, the flesh of species from sections Uracei and Brunnei is dark brown at least at the base of the stipe, the lamellae are dark brown when old, and the exsiccatae are blackish. Cortinarius biformis Fr. has paler lamellae, paler pileus or when the pileus is darker the stipe has bluish tints, and paler exsiccatae. Notes Cortinarius badiolaevis 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. But whereas the species-level taxonomy seems to be clear, the relationships to other species need further study. No close relatives of the species are known so

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far. In the MP analysis, C. badiolaevis sometimes clustered together with C. malachius but with a low (<50%) support value. In addition, it does not seem to be closely related to any of the morphologically similar species. Collections examined Spain. Barcelona: Figols (Berguedà) 1600 m, under Pinus sylvestris in calcareous soil, 30 Sept 2005, J. Ballarà & R. Mahiques MES-4175 (Mahiques pers. herb., HM017848). Castelló: Morella, Mas de la Carcellera, under Pinus nigra, in calcareous soil, 26 Oct 2005, K. Liimatainen & T. Niskanen 05-266 (H, HM017847). Sweden. Upland: Närke, Lillkyrka parish, Ekebergsbrottet, mesic coniferous forest (Picea, Pinus) in calcareous soil, 26 Sept 2004, I. Kytövuori, K. Liimatainen & T. Niskanen F04960 (holotype H, isotype NY, HM017846). Ångermanland: Säbrå, Hårsta, in blueberry spruce forest (Picea), 6 Oct 1993, H. Lindström et al. CFP 1251 (S, HM017845).

Acknowledgements We thank André Bidaud and Håkan Lindström for the taxonomical comments and revision of the manuscript, Tess Barlow for revision of English, Tor Erik Brandrud, Håkan Lindström, Hans Marklund, Jacques Melot and Siw Muskos for lending the material, Heino Vänskä for revision of the Latin description, and referees for valuable comments. This work was supported by the Swedish Taxonomy Initiative (dha 165/08 1.4) and Academy of Finland (project number 129052).

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