Mycol Progress DOI 10.1007/s11557-016-1264-y

ORIGINAL ARTICLE

Cortinarius stjernegaardii and C. kristinae (Basidiomycota, Agaricales), two new European species with a mainly northern distribution Tobias Guldberg Frøslev 1 & Tor Erik Brandrud 2 & Bálint Dima 3,4

Received: 8 November 2016 / Revised: 15 December 2016 / Accepted: 19 December 2016 # German Mycological Society and Springer-Verlag Berlin Heidelberg 2017

Abstract We describe two new, mainly North European species of basidiomycetous fungi. Cortinarius stjernegaardii belongs to the section Percomes containing several similar species with greenish-yellow, anthraquinonoid pigments and peculiar smells. The species has hitherto been identified as^ C. bulbopodius^ in the Nordic countries, an epithet which, however, is reduced to a synonym of C. aurilicis. Cortinarius kristinae belongs to the section Calochroi, which includes many morphologically similar species with sharply marginate bulbose stipes and yellow pileus colours. The species are distinguished from related taxa by molecular data (ribosomal ITS region), and typical specimens can be identified by a combination of basidiocarp coloration, stature, microscopy, reactions with KOH, and habitat.

Keywords Calochroi . Cortinariaceae . Europe . ITS . Percomes . Phylogeny . Taxonomy

Section Editor: Zhu-Liang Yang * Bálint Dima [email protected] 1

Department of Biology, University of Copenhagen, Universitetsparken 15, 2100 København Ø, Denmark

2

Norwegian Institute for Nature Research (NINA), Gaustadalléen 21, 0349 Oslo, Norway

3

Department of Plant Anatomy, Institute of Biology, Eötvös Loránd University, Pázmány Péter sétány 1/c, H-1117 Budapest, Hungary

4

Department of Biosciences, Plant Biology, University of Helsinki, P.O. Box 65, FI-00014 Helsinki, Finland

Introduction Cortinarius is the largest genus in the Agaricales (Fungi), which includes most of the well-known mushroom-forming species of basidiomycetes. The taxonomy of European species of Cortinarius have been treated relatively intensively in the last two decades, and the use of morphological studies along with molecular phylogenetic analyses primarily of the ITS (internal transcribed spacer region of the ribosomal cistron) region of the ribosomal DNA has resulted in a much better understanding of the species diversity and relevance of morphological characters for species delimitation and identification (e.g. Frøslev et al. 2007; Brandrud et al. 2014b; Liimatainen et al. 2014). Both species studied here are traditionally treated in the morphologically circumscribed subgenus Phlegmacium, which encompasses taxa with dry stipe and viscid pileus. Although we are now much closer to a common understanding of the European species in Cortinarius subgenus Phlegmacium (Jeppesen et al. 2012), there still remains work with stabilising the nomenclature and evaluating the species level taxonomy of some of the less studied groups. In the process of re-evaluating species limits and the application of names in the subgenus Phlegmacium by studies of morphology and molecular markers, we discovered two undescribed species which we have known for quite a while. One of the species has hitherto been called C. bulbopodius (Chevassut & Rob. Henry) Bidaud & Reumaux (= C. nanceiensis var. bulbopodius Chevassut & Rob. Henry) in the nordic countries (see, e.g., Brandrud et al. 1996, 2010). However, investigation of ITS data from the type material of C. bulbopodius made it evident that it was identical to C. aurilicis Chevassut & Trescol. This left our species without a formal name. Therefore, we here describe it as Cortinarius stjernegaardii, and chose to name it after our good friend and colleague, Thomas Stjernegaard Jeppesen.

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The second species has also been known to us for many years, and was preliminary introduced without a valid diagnosis in Funga Nordica (Jeppesen et al. 2012). It belongs to the Calochroi lineage with numerous species that are very difficult to separate based on morphology, but where the combination of molecular markers and careful re-evaluation of morphological characters has made it possible to distinguish many species reliably (Frøslev et al. 2007). The species is named after the wife of the second author.

Materials and methods Morphological study The taxonomic descriptions are based on material studied by the authors. The measurements of macro-morphological characters are based on expanded, but never old (and then often aberrant), basidiocarps. Macrochemical reagents applied were 2% and 10–40% KOH. The terminology of characters follows Brandrud et al. (1990) with minor adjustments (see Brandrud 1996). Microscopical structures were observed partly from fresh material mounted in H2O, often with a drop of 40% KOH subsequently added, and partly from dried material mounted in H2O and then KOH. The descriptions of the pigment topography are based largely on observations from H2O mounts of fresh, preferentially young material. The spores were studied and measured in 2% KOH, with a ×100 oil immersion lens. From each basidiocarp, a random selection of mature spores obtained from cortina remnants were measured excluding apiculus and ornamentation. Young, immature basidiocarps were avoided. Ten spores were measured per specimen. Mean values (MV) of spore length and width as well as Q values (length/width ratio) were then calculated for each specimen. MV were calculated for each species. The pileipellis was studied from radial (longitudinal), freehand sections, preferentially from fresh material. Wedge-like sections, ultrathin at the one end, were obtained by cutting at a slightly oblique angle. The sections were cut from approx. 5 × 5 × 3 mm large pieces taken from young but expanded pilei, and at a position midway to the centre. Herbarium acronyms follow Thiers (continuously updated). The following acronyms are used for collectors and in collection numbers: TEB = Tor Erik Brandrud, TSJ = Thomas Stjernegaard Jeppesen and TF = Tobias Guldberg Frøslev. All material is kept at the herbarium of Oslo (O) unless otherwise noted. Molecular study After morphological studies, the internal transcribed spacer (ITS) of eleven specimens of C. stjernegaardii, six specimens of C. kristinae, the type specimen of C. bulbopodius, three specimens o f C. aurilicis, and one specimen of

C. nanceiensis, were sequenced (Table 1.) following the DNA extraction methods and PCR procedures published by Frøslev et al. (2007, 2015). The primer pairs ITS1F/ITS4B or ITS5/ITS4 (White et al. 1990; Gardes and Bruns 1993) were used in the polymerase chain reactions (PCR). Sequencing was carried out at Macrogen (Amsterdam, The Netherlands) and LGC Genomics (Berlin, Germany) with the same primers as those applied in PCR. The chromatograms were checked and edited with CodonCodeAligner 4.1. (CodonCode, Centerville, MA, U.S.A). BLAST queries of the public databases (GenBank: http://www.ncbi.nlm.nih.gov/ and UNITE: http://unite.ut.ee/) were used to check for similar sequences. Based on the BLAST results, we compiled a dataset using ITS region in addition to the D1/D2 regions of the large subunit (LSU) of genetically related species and morphological similar taxa to show their relationship compared to the new species (Table 1). Multiple sequence alignment was done with MAFFT (online version 7) with use of the L-INS-i strategy (Katoh and Standley 2013). Manually adjustments were done in SeaView (Gouy et al. 2010). Gaps were treated as missing data. The alignment is deposited in TreeBASE under TB2:S20162 (http://purl.org/phylo/treebase/phylows/study/). Maximum likelihood (ML) phylogenetic analyses were performed with PhyML 3.0 (Guindon et al. 2010), using the GTR+I+Γ model of evolution. Branch support was tested using the non-parametric Shimodaira–Hasegawa version of the approximate likelihood-ratio test (SH-aLRT), implemented in PhyML (Anisimova et al. 2011; Bellanger et al. 2015). The ML phylogenetic tree was visualised and edited in MEGA6 (Tamura et al. 2013).

Results Phylogenetic analyses The combined ITS and LSU dataset comprised 63 taxa (41 from GenBank and UNITE) and 1355 characters. The 22 newly generated sequences were submitted to GenBank and the accession numbers are available in Table 1. The bestscoring ML tree from our PhyML analyses is shown in Fig. 1. Bootstrap (BS) values over 80% was considered as strong statistical branch support. Our analysis resolved the focused sections referred to as /Percomes (BS = 95%) and /Calochroi (BS = 100%). The two new species proposed here, Cortinarius stjernegaardii and C. kristinae, fall within the sections Percomes and Calochroi, respectively, as strongly supported terminal clades (BS = 99% each). Cortinarius stjernegaardii forms a strongly supported (BS = 97%) sister clade to C. aurilicis. The dissimilarity between these two species is 6% (>30 substitutions and indel

Mycol Progress Table 1

Cortinarius sequences used in the phylogenetic analysis

Species

Voucher

Country

Sequence origin

Accession no.

Notes

C. aurilicis Chevassut & Trescol

TSJ 1998–101

France

Frøslev et al. (2005)

DQ083772

C. aurilicis

Henry 70671

France

This study

KY315438

Holotype of C. nanceiensis var. bulbopodius

C. aurilicis

TUB 011865

Germany

Garnica et al. (2005)

AY669520

As C. nanceiensis var. bulbopodius

C. aurilicis

TUB 019807

Germany

Garnica et al. (2016)

C. aurilicis C. aurilicis

Huth 17–09–2010 Münzmay 44/07

Germany This study Germany This study

KY315439 KY315436

KJ421100

C. aurilicis

TEB 445–10

Germany This study

KY315437

C. bergeronii (Melot) Melot

TUB 012698

Italy

Garnica et al. (2009)

EU057048

As C. cedretorum var. suberetorum

C. caerulescens (Schaeff.) Fr.

CFP 853

Belgium

KF732271

Epitype

C. caesiocortinatus Jul. Schäff.

TSJ 2002–028

Liimatainen et al. (2014) Frøslev et al. (2005)

C. corrosus Fr. C. corrosus

Czech Rep. IB 19880210 Italy Arangu-Cort-01102201 Spain

DQ083774

Peintner et al. (2001) AF325618 Garnica et al. (2009) EU057057

C. corrosus C. crassus Fr. C. dalecarlicus Brandrud C. dalecarlicus C. dionysae Rob. Henry C. elegantissimus Rob. Henry

TUB 020429 TUB 019696 TAAM 128 TF2000-108 TUB 020423 TUB 012710

Germany Germany Estonia Italy Germany Germany

Garnica et al. (2016) Garnica et al. (2005) UNITE Frøslev et al. (2007) Garnica et al. (2016) Garnica et al. (2009)

KJ421199 AY669544 UDB015931 DQ663284 KJ421135 EU057037

C. flavipallens Kytöv., Liimat. & Niskanen C. flavipallens

H 6032745

Finland

KF732554

TK 368

Finland

C. glaucopus (Schaeff.) Fr.

CFP 786

Sweden

C. kristinae sp. nov. C. kristinae

EB 211/09 TEB 227–06

Norway Norway

Liimatainen et al. (2014) Liimatainen et al. (2014) Liimatainen et al. (2014) This study This study

C. kristinae C. kristinae C. kristinae C. kristinae C. laberiae Münzmay, B. Oertel & Saar

TEB 441–04 KS-CO1930 KS-CO2061 TEB 225–07 TUB 01189

Norway Sweden Sweden Sweden Germany

This study This study This study This study Garnica et al. (2005)

KY315418 KY315421 KY315420 KY315419 AY669563

Holotype

C. laberiae C. metarius Kauffman

TUB 011658 PML 228

Germany France

Garnica et al. (2009) Frøslev et al. (2007)

EU655663 DQ323959

Holotype Holotype of C. barbarorum

Holotype

KF732556 KF732315

Neotype

KY315422 KY315423

As C. subarquatus

C. metarius

TUB 011648

Germany

Garnica et al. (2009)

EU655671

As C. barbarorum

C. metarius

MICH 10374

U.S.A.

KF732347

Holotype

C. nanceiensis Maire C. nanceiensis

TU 105337 TUB 011389

Estonia Germany

Liimatainen et al. (2014) UNITE Garnica et al. (2003)

C. nanceiensis C. nanceiensis C. nanceiensis C. nanceiensis C. olympianus A.H. Sm.

TUB 011422 TUB 019742 TUB 019804 TEB 278–10 IK 94–1225

Germany Germany Germany Norway Finland

C. olympianus

MICH 10386

U.S.A.

Garnica et al. (2003) Garnica et al. (2016) Garnica et al. (2016) This study Liimatainen et al. (2014) Liimatainen et al. (2014)

UDB018631 AY174855 AY174856 KJ420982 KJ421097 KY315435 KF732553 KF732364

Holotype

Mycol Progress Table 1 (continued) Species

Voucher

Country

Sequence origin

Accession no.

Notes

C. percomis Fr.

TN 08–041

Finland

KF732380

Neotype

C. percomis

CFP 1104

France

C. percomis C. piceae Frøslev, T.S. Jeppesen & Brandrud C. piceae C. piceae

TUB 011868 TUB 012691

Germany Germany

Liimatainen et al. (2014) Liimatainen et al. (2014) Garnica et al. (2005) Garnica et al. (2009)

TF 2004 026 CFP 508

Sweden Sweden

Frøslev et al. (2007) Frøslev et al. (2007)

DQ663378 DQ663380

C. prasinus (Schaeff.) Fr.

TUB 011431

Germany

Garnica et al. (2003)

AY174835

C. purpurascens Fr.

TUB 01140

Germany

Garnica et al. (2016)

KJ421138

C. russeoides M.M. Moser

TU 106566

Estonia

UNITE

UDB011259

C. russeoides C. russeoides

KS-CO1000 CFP 520

Sweden Sweden

Garnica et al. (2016) Garnica et al. (2016)

KJ421060 KJ421210

C. stjernegaardii sp. nov. C. stjernegaardii

Saar 19–10–2012 O-F-75463

Germany This study Norway This study

KY315427 KY315430

C. stjernegaardii C. stjernegaardii C. stjernegaardii C. stjernegaardii C. stjernegaardii C. stjernegaardii C. stjernegaardii

O-F-75604 TEB 212–10 TEB 355–10 TEB 355–14 TEB 395–08 TEB 422–08 TEB 737–13

Norway Norway Norway Norway Norway Norway Norway

This study This study This study This study This study This study This study

KY315429 KY315428 KY315434 KY315425 KY315433 KY315432 KY315424

C. stjernegaardii C. stjernegaardii

CFP 1216 TEB 1099–11

Sweden Sweden

This study This study

KY315431 KY315426

C. variecolor (Pers.) Fr.

TUB 011462

Germany

Garnica et al. (2003)

AY174796

KF732520 AY669529 EU056956 Holotype As C. calochrous var. coniferarum

Holotype

Newly generated sequences are in bold

positions) in the ITS region which is much more pronounced than the intraspecific genetic variability detected among the sequences of C. stjernegaardii (<1%; 0 to 5 substitution and indel differences). This species was formerly often misinterpreted as C. nanceiensis var. bulbopodius, but the ITS sequence of the holotype specimen appeared to be identical to C. aurilicis (Fig. 1). The phylogenetic position of C. kristinae within the section Calochroi has not yet been fully resolved in our analysis. The species falls within a well-supported subclade showing a polytomy, where some morphological similar species (e.g. C. flavipallens Kytöv., Liimat. & Niskanen, C. dalecarlicus Brandrud) also belong to. Using BLASTn searches of public databases, we attempted to identify the possible closest relative of C. kristiniae in terms of nucleotide difference. Cortinarius olympianus A.H. Sm., a member of the same

subclade where C. kristinae does belong to, appeared to have the most similar ITS sequence with 2% dissimilarity in the ITS region (15 substitution and indel position differences). All sequenced specimen of C. kristinae possessed identical ITS.

Taxonomy Cortinarius stjernegaardii Brandrud & Frøslev, spec. nov. MycoBank: MB814401 Fig. 2 Etymology: named after our colleague Thomas Stjernegaard Jeppesen. Holotype: Sweden, Gotland, Ekstakusten, with Corylus avellana, 29 Sept 2011, Tobias Guldberg Frøslev, Thomas Stjernegaard Jeppesen & Tor Erik Brandrud, TEB 1099–11 (O). GenBank: KY315426

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85

TEB422-08 O-F-75604 TEB355-10 O-F-75463 TEB1099-11 (holotype) TEB355-14 Cortinarius stjernegaardii Saar 19-10-2012 99 TEB395-08 TEB737-13 CFP1216 97 TEB212-10 TEB445-10 KJ421100 C. aurilicis Henry 70671 AY669520 C. nanceiensis var. bulbopodius Cortinarius aurilicis /Percomes 99 DQ083772 C. aurilicis 100 Huth 17-09-2010 Münzmay 44/07 AY174855 C. nanceiensis KJ420982 C. nanceiensis TEB278-10 Cortinarius nanceiensis 97 AY174856 C. nanceiensis KJ421097 C. nanceiensis UDB018631 C. nanceiensis 95 KF732520 C. percomis AY669529 C. percomis Cortinarius percomis 100 KF732380 C. percomis KJ421060 C. russeoides KJ421210 C. russeoides Cortinarius russeoides 100 UDB011259 C. nanceiensis AY174796 C. variicolor DQ083774 C. caesiocortinatus KF732271 C. caerulescens KJ421135 C. dionysae KF732315 C. glaucopus AY174835 C. prasinus EU057048 C. bergeronii EU057037 C. elegantissimus 100 99 KF732347 C. metarius DQ323959 C. barbarorum Cortinarius metarius EU655671 C. barbarorum 100 EU655663 C. laberiae Cortinarius laberiae AY669563 C. subarquatus 83 AF325618 C. corrosus 99 KJ421199 C. corrosus Cortinarius corrosus EU057057 C. corrosus 84 99 KF732556 C. flavipallens Cortinarius flavipallens KF732554 C. flavipallens /Calochroi EB211/09 TEB441-04 (holotype) 99 TEB227-06 Cortinarius kristinae TEB225-07 KS-CO2061 90 KS-CO1930 100 DQ663284 C. dalecarlicus Cortinarius dalecarlicus UDB015931 C. dalecarlicus 94 KF732364 C. olympianus Cortinarius olympianus 85 KF732553 C. olympianus EU056956 C. piceae DQ663380 C. calochrous var. coniferarum Cortinarius piceae 99 DQ663378 C. piceae KJ421138 C. purpurascens AY669544 C. crassus

0.01

Fig. 1 Maximum likelihood phylogram of the sections Percomes and Calochroi within the genus Cortinarius inferred from PhyML analysis of nrDNA ITS + LSU D1/D2 sequences focusing on the morphological similar species to C. stjernegaardii and C. kristinae. Cortinarius crassus served as outgroup. Members of section Percomes are marked in red

whereas those of section Calochroi are in blue. Newly generated sequences are highlighted in bold. Numbers at branches represent bootstrap percentages (only support values above 80% are shown). Bar indicates 0.01 expected change per site per branch

Pileus 40–90(−110) mm, (hemi-)spherical, then plano-convex, sometimes slightly umbonate; fairly bright and persistently greenish-yellow towards margin,

centre when young dark greyish-brown to olive-brown, with age more red-brown, and then ochre-brown at margin.

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Fig. 2 Cortinarius stjernegaardii sp. nov. Holotype (TEB 1099–11). Sweden, Gotland, Ekstakusten, 29 Sept 2011. Photograph: Tobias Guldberg Frøslev

Lamellae wax yellow to greyish-yellow, rarely with a faint bluish tinge when very young. Stipe 40–90 × 8–20(−30) mm, with a fairly distinctly marginate bulb (40 mm), greenish-yellow as pileus margin, apex sometimes with a bluish reflex when very young. Universal veil fairly abundant at or just above bulb margin, olive-grey to red-brown. Basal mycelium whitish with ochre yellow to saffron yellow mycelial strands. Context pale yellowish-green in pileus and bulb when very young, later almost white, brighter greyish-green to greenishyellow in stipe, especially towards surface. Odour of unripe banana when cut. Macrochemical reactions: 2% and 40% KOH pale reddish in the context, more strongly blood-red in the bulb. Basidiospores 11–13 × 6.5–7.5 μm (MV = 11.97 × 7.12 μm), Qav = 1.68, faintly citriform to amygdaliform, very distinctly and fairly coarsely verrucose, apex with a lense-like, thickened wall, suprahilar plage fairly distinct. Basidia 8–11 μm wide. Pileipellis simplex, thick, with scattered to sometimes fairly abundant extracellular, greenish-yellow pigment; encrustation weak or sometimes absent. Extractable pigments: The species contains the greenishyellow phlegmacin-8′-methylether (Brandrud 1998). Habitat and distribution: The species is found mainly in boreo-nemoral frondose forests, once also in a nemoral (temperate) site. At present, it is known from 20 localities/ sites in the Oslofjord district of SE Norway (Brandrud et al. 2011, 2014a as C. bulbopodius), and 20 sites in C/SE Sweden (Kinnekulle S of Vänern, Öland and Gotland; T. Knutsson, M. Krikorev, personal communication). Furthermore, it has been found once in the Rhine valley district of Germany. It is associated with Tilia cordata and Corylus avellana, possibly also Quercus robur, in dry, calcareous shallow soil; in Norway

(Oslofjord) and Sweden (Vänern S), it mainly occurs in calcareous lime forests, whereas on Öland–Gotland also in hazel thickets or hazel–oak woodlands (Brandrud 1999; Brandrud and Bendiksen 2001; Brandrud et al. 1996, as C. nanceiensis var. bulbopodius, Brandrud et al. 2011, as C. bulbopodius). In Germany, the species was found under Tilia cordata and Carpinus betulus on rich loess soil. The species is rare, possibly of relict nature (Brandrud 1999 as C. nanceiensis var. bulbopodius). Comments: Cortinarius stjernegaardii is characterised by its bicoloured pileus with a greenish-yellow margin and an olivaceous (red)brown centre, a more or less marginate bulbous stipe base, and a smell of banana or apples. The species differs from the often co-occurring C. nanceiensis Maire in its more distinctly marginate bulb and stronger and more persistently greenish-yellow colours on stipe, pileus margin and in the context. Cortinarius nanceiensis is furthermore somewhat warmer reddish-brown in colour on the veil at the bulb and on the pileus centre. When very young, C. stjernegaardii sometimes has a faint bluish-green tinge in the context and on the lamellae; this is never seen in C. nanceiensis. With age, C. stjernegaardii may develop warmer pileus colours, and can then be difficult to distinguish from C. nanceiensis. The two species have very similar microcharacters, the spores being slightly larger and the extracellular pigment being slightly more abundant in C. stjernegaardii. The taxa have different distributions as C. stjernegaardii seems to be more or less restricted to the boreonemoral zone of Fennoscandia, whereas C. nanceiensis has a wide distribution in calcareous districts in the whole Europe, especially in montane Fagus–Abies forests. The species have overlapping distributions and ecology in calcareous Tilia–Corylus and Quercus–Corylus forests of southern Scandinavia. Cortinarius stjernegaardii is very similar to its sister species C. aurilicis. However, C. aurilicis is a southern, preferentially Mediterranean species with outposts in northern Germany (Brandrud and Schmidt-Stohn 2011), and their distributions do not seem to overlap. Cortinarius stjernegaardii is distinguished from C. aurilicis by its normally smaller, more slender basidiocarps and is less greenish pigmented in context and on lamellae. Cortinarius aurilicis is normally more red-brown in the pileus (centre) (cf. the name Baurilicis^) than our species, but the colours may overlap (Brandrud and Schmidt-Stohn 2011). Cortinarius stjernegaardii can also be confused with a number of distantly related, abruptly bulbous frondose forest species from section Fulvi, such as C. bergeronii (Melot) Melot, C. elegantissimus Rob. Henry and C. prasinus (Schaeff.) Fr. However, C. stjernegaardii differs from them in its stronger greenish-yellow context, olive-grey to redbrown veil remnants just above the bulb margin, a fruit-like smell, and a weaker (negative to red-brown) KOH reaction. The spores of C. stjernegaardii are larger than those of

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C. bergeronii and C. prasinus, and smaller than those of C. elegantissimus. Cortinarius stjernegaardii has for some time been identified as C. bulbopodius or C. nanceiensis var. bulbopodius in the Nordic countries (see, e.g., Brandrud et al. 1996, 2010; Jeppesen et al. 2012), but ITS sequence data from the type material of C. bulbopodius has shown this to be a synonym for the southern/Mediterranean C. aurilicis (Fig. 1). This synonymy was already indicated in Jeppesen et al. (2012), where our species was treated as C. bulbopodius s. Brandrud. Additional specimens examined (*including macrocharacters): Germany. Baden-Württemberg, Kaiserstuhl, under Tilia and Carpinus, G. Saar 19–10–2012. Norway. Oslo, Bygdøy, Dronningberget, TEB 505–80*, TEB 201–82*, TEB 74– 85*, TEB 63–88*, Bygdøy, Reinsdyrlia, TEB 561–04, TEB 527–11, O-F-75604, Bygdøy, Hengsåsen, TEB 603–04; Akershus, Bærum, Løkkeåsen, TEB 150–85*, TEB 151–01, TEB 781–11, Kalvøya, TEB 422–08*; Asker, Spireodden, TEB 83–94*, TEB 395–08*, TEB 212–10*, Nesøytjern nature reserve, TEB 1032–11*, TEB 1036–11, Sjøstrandveien, TEB 335–10*, TEB 1044b-11, Elnestangen NR, B. Dima & TEB 408–15; Buskerud, Røyken, Slemmestad, Tåje, TEB 355–10*, TEB 823–11*, R. Braathen & E.W. Hanssen, O-F75463, Bøsnipa, TEB 815–11*, B. Dima & TEB 737–13*, Slemmestadveien, B. Dima & TEB 512–15; Hole, Nes, TEB 156–85, TEB 162–85*, TEB 102–97, TEB 738–11, Aurdalsvingen, TEB 786–11*, Bråtafjellet, TEB 704–11, TEB 711–11; Oppland, Biri, Eriksrud, TEB 172–85*, 173– 85*, 75–88*, 261–00, B. Dima & TEB 355–14, TEB 348–15, TEB 653–15; Telemark, Porsgrunn, Brattås, TEB 874–11, TEB 934–11. Sweden. Västergötland, Kinnekulle, Medelplana, Råbäck, TEB 112–93*, CFP 1216*, S. Jacobsson 79141 (as C. percomis, GB); Öland, Långlöt, Åstad, TEB 21–96*; Gotland, Fröjel, Ekstadkusten nature reserve, B. Wasstorp, TEB 1094–11. Cortinarius kristinae Brandrud, spec. nov. MycoBank: MB814403 Fig. 3 Etymology: named after Kristin H. Brandrud. Holotype: Norway, Oppland, Lunner, under Picea abies, 14 Sept 2004, Tor Erik Brandrud, TEB 441–04 (O; isotype, C). GenBank: KY315418. Pileus 40–100 mm, (hemi-)spherical, then plano-convex, glutinous, glabrous, often appearing almost tomentose to fibrillose when dry, margin often more or less persistently involute; involute margin sometimes with small slime drops; often relatively uniformly coloured, ochraceous-yellow to ochraceous-brownish, often with a more distinct yellow tinge, sometimes with darker spots, involute margin significantly

Fig. 3 Cortinarius kristinae sp. nov. Holotype (TEB 441–04). Norway, Oppland, Lunner, 14 Sept 2004. Photograph: Tor Erik Brandrud

paler, yellowish to cream, margin sometimes finely fibrillose from cortina remnants, in damage (snails, insects) often somewhat saffron discoloured; universal veil often seen on the pileus as diffuse small scales which become brownish at centre. Lamellae emarginate, greyish-white, edge paler, often fimbriate. Stipe 40–80(−100) × 10–20 mm, glabrous, shining, with an abruptly marginate bulb (up to 35 mm wide), whitish-cream, sometimes staining somewhat ochraceous to brownish; bulbipellis white, mycelial strands whitish. Universal veil on the bulb margin usually fairly abundant, ochraceous to more yellow. Cortina relatively abundant, with age heavily covered with rust-brown spore powder. Context whitish to cream, sometimes with greyish hygrophaneous spots/stripes in stipe (apex), often spotted ochraceous with age. Smell indistinctly phlegmacioid to distinctly earth-like. Taste indistinct. Macrochemical reactions: 10–40% KOH on pileus bloodred, darker on most coloured parts, on bulbipellis normally distinctly pink, on bulb margin (universal veil remnants) similar to pileus, in context negative to pale grey-rose. Basidiospores 10–12 × 5.5–6.5 μm (MV = 10.9 × 6.3 μm), Qav = 1.8, acutely amygdaloid to citriform with narrow extended apex (often almost mammiform, often with distinct ventral constriction at apex); citriform (to fusiform) in frontal view; coarsely verrucose, warts relatively small. Pileipellis simplex, hyphae hyaline to yellowish, hyphae filled with relatively abundant yellow necropigment; ±hyaline crystals present both extra- and intracellularly. Microchemical reactions (10% KOH): pileipellis hyphae with necropigment brown to slightly vinaceous, ±hyaline hyphae pinkish, crystals pink; bulbipellis hyphae pink. Habitat and distribution: Boreal and boreonemoral coniferous forests. Associated with Picea on richer to ± calcareous soils, but not found together with the strictly calciphilous species. Also found in younger Picea plantations. Apparently very rare, and apparently more or less northern; so far known from northern

Mycol Progress

Europe (Norway and Sweden), with an oceanic tendency: comparatively many finds in humid, west-coast spruce forests of boreal rain forest character in Norway. Recently, the species has also been verified from sequence data from France (A. Bidaud, personal communication). Comments: Cortinarius kristinae is a typical representative of the large section Calochroi, characterised by the combination of a simplex pileipellis, a coarse, almost net-like ornamentation of the basidiospores, and an almost smooth, not fibrillose pileus. It is distinguished from other species of section Calochroi by the combination of a yellow pileus, yellow universal veil, pinkish-red alkaline reaction on bulbipellis and pileus and an absence of violaceous colours. The most similar species are C. corrosus Fr., C. dalecarlicus, C. flavipallens, C. laberiae Münzmay, B. Oertel & Saar, C. metarius Kauffman, and C. piceae Frøslev, T.S. Jeppesen & Brandrud. Cortinarius corrosus and C. dalecarlicus have the initially greyish white lamellae in common with our C. kristinae, but lack the vivid yellow pileus colour and the red KOH reaction. Cortinarius flavipallens also lacks clear and distinct yellow coloration of the pileus and universal veil, and seems to have more greyish (even bluish grey?) lamellae when young (Liimatainen et al. 2014). Cortinarius laberiae shows weaker alkaline reactions, with the pileus being more red-brown and the bulbipellis only weakly and slowly pinkish. Furthermore, the species seems to be associated with Abies on strictly calcareous ground (Münzmay et al. 2009). Cortinarius piceae might in some stages look very similar to C. kristinae, but differs in a normally darker-brown, more appressed scaly pileus with a brownish KOH reaction, initially pale lilac lamellae, slightly smaller spores and a negative KOH reaction on the bulbipellis. Cortinarius metarius always has a distinct lilac-violaceous tinge on the lamellae (and often also on stipe apex), but otherwise shows overlap, e.g., in pileus colours, in spore size and the bright pink KOH reaction on the bulbipellis.

Additional specimens examined (*including macrocharacters): Norway. Hedmark, Ringsaker, Nes, TEB 64–92*; Åmodt, Gråfjell, TEB & E. Bendiksen, TEB-EB 90–00*; Hordaland, Ulvik, Lindebrekke, TEB 216–06*, Voss, Malahaugen S, TEB 227–06*, TEB 228–06*, TEB 232–06*; Oppland, Lunner, S Oppdalen, TEB 17–90*, TSJ & TEB, TEB 500– 04*, Lunner, Råstad, TEB 134–93*, Gjøvik, Biri, TEB 47– 92*; Oslo, Bogstad, TEB 176–83*; Buskerud, Ringerike, Øyangen, TEB 505–13*, TEB 506–13*; Sogn og Fjordane, Sogndal, Kaupang centre, TEB 254–00*; Nord-Trøndelag, Steinkjer, Skrattåsen, L. Nagy (no. 322), T. von Bonsdorff (H), Byhalla, A.K. Wollan & K. Høiland (no. 258); Møre og Romsdal, Rindal, Svorklia Ø, E. Bendiksen, EB 211/09. Sweden. Dalarna, Mora, N of Rädbjörka, I. Kytövuori, TEB

225–07, Alderängarna, Karl Soop, KS-CO1930 (S), KSCO2061 (S). Acknowledgements Several persons and institutions have been involved in sequencing of the studied material. We thank Mika Bendiksby and Ingeborg Bjorvand Engh (University of Oslo), Gábor M. Kovács (Eötvös Loránd University, Budapest), Martin Unterseher (University of Greifswald), Ali Tahir and Marco Thines (Goethe University, Frankfurt am Main and Senckenberg Biodiversity and Climate Research Centre) for contributing with ITS sequences. The Cortinarius, Flora Photographica team, the JEC-DNA Phlegmacium Group, Egil Bendiksen (NINA, Oslo) and Thomas Stjernegaard Jeppesen (Copenhagen), are thanked for cooperation in the study of the species. Furthermore, we thank all the mycologists who have provided material for molecular or morphological study. Part of the data presented here are the results of a Species Project on Cortinarius 2010–2012, funded by The Norwegian Biodiversity Information Centre. Much data on C. stjernegaardii have also been obtained through a study on calcareous Tilia forests, as part of a national program for mapping and monitoring Red-listed species (the ARKO project), funded through the Norwegian Environment Agency. We thank the curator of PC (Bart Buyck) for managing the loan of the type specimen of Cortinarius nanceiensis var. bulbopodius.

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