Plant Syst Evol DOI 10.1007/s00606-016-1349-8

ORIGINAL ARTICLE

Galerina saxicola (Fungi, Agaricales) is conspecific with G. stordalii and new data on ecology of the latter species Jan Holec1

•

Martina Vasˇutova´2 • Miroslav Kolarˇ´ık3 • Martin Krˇ´ızˇ1

Received: 24 May 2016 / Accepted: 18 August 2016 Ó Springer-Verlag Wien 2016

Abstract The holotype of Galerina saxicola Svrcˇek, a never revised species described from the Czech Republic in 1994, was studied in detail. Due to its poor state and the impossibility to obtain DNA data, we fixed its taxonomic position by designating an epitype using recent material from the type locality. The species proved to be conspecific with Norwegian and Czech collections of Galerina stordalii A.H.Sm., both morphologically and molecularly. Full synonymy and diagnostic characters of G. stordalii are provided, its morphological and ecological variability is discussed, and information on type specimens is corrected. Our collections document that G. stordalii has a broader ecological amplitude than thought before, living not only on Sphagnum and peat in boreal and arctic-alpine habitats like bogs and snow beds, but also among mosses in boggy spruce forests, on decaying conifer trunks in old-growth forests and on moist sandstone rocks in a river canyon under the influence of climatic inversion. Keywords Ecology  Epitypification  Europe  Hymenogastraceae  Morphology  Phylogeny

Handling Editor: Stephen W Peterson. & Jan Holec [email protected] 1

Mycological Department, National Museum, Cirkusova´ 1740, Praha 9, Czech Republic

2

Department of Botany, Faculty of Science, University of South Bohemia, Branisˇovska´ 31, Cˇeske´ Budeˇjovice, Czech Republic

3

Institute of Microbiology ASCR, Vı´denˇska´ 1083, 142 20 Praha 4, Czech Republic

Abbreviations L number of lamellae reaching the stipe l number of lamellulae between each pair of lamellae Q spore length/width quotient Qav mean of Q

Introduction The genus Galerina Earle (Basidiomycota, Agaricales) belongs to the Hymenogastraceae family (Matheny et al. 2006) and comprises about 250 species worldwide (Kirk et al. 2008). However, 365 Galerina names are included in the Mycobank database (www.mycobank.org, accessed 27 April 2016) as legitimate names. According to Index fungorum (www.indexfungorum.org, accessed 27 April 2016), 50 of them are considered synonyms, which is obviously not a final number. The first and so far only detailed phylogenetic study of Galerina strongly suggests that the genus is polyphyletic (Gulden et al. 2005) and should be segregated into at least three monophyletic genera. Nevertheless, verification of this finding using multiple unlinked loci nor formal taxonomic changes has yet been completed. Morphological study of most Galerina species is complicated due to their small basidiomata and insufficiently known variability of macro- and microcharacters. Many species are known from their type locality only (Smith and Singer 1964), and types are often poorly preserved or lacking. Interpretation of such species is very difficult or even impossible. As already mentioned by Matheny et al. (2015), a revision of poorly documented taxa and an elucidation of their phylogenetic affinities is one of the challenging tasks in current taxonomy in an effort to assess the real diversity and evolution of Agaricales.

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In Northern Europe, the best investigated region of Europe concerning Galerina taxonomy and distribution, 50 Galerina species are reported in the latest overview (Gulden 2012), which strongly contrasts with the seven times higher number of legitimate names (see above). In this situation, profound revision of all published names is highly desirable to correctly delimit the really existing taxa and clear up their synonymy. One of the hitherto unrevised taxa is Galerina saxicola Svrcˇek, lately described from the Kamenice river canyon in North Bohemia, Czech Republic (Svrcˇek 1994). The fungus was collected on moist sandstone rocks covered by foliose liverworts and mosses. Its key characters should be the yellow-brown pileus lacking veil, scarce lamellae, white and pruinose stipe, absence of clamps, finely ornamented spores with small pore, and lageniform cheilocystidia with subcapitate apex. At present, the type locality represents the first, strictly protected zone of the Cˇeske´ Sˇvy´carsko (Bohemian Switzerland) National Park. Its geology, geomorphology and habitats are described by Ha¨rtel et al. (2007). According to Svrcˇek (1994), G. saxicola belongs to Galerina subg. Tubariopsis (Ku¨hner) A.H.Sm. & Singer. He compared it especially with G. stordalii A.H.Sm., a boreal to arctic-alpine species growing on Sphagnum and possessing clamps (Smith and Singer 1964; Gulden et al. 1985). Galerina saxicola is not included in any monographs of Galerina (e.g. De Haan and Walleyn 2002, 2006, 2009; Gulden et al. 2005; Gulden 2012), and no sequence is present in GenBank. Rich material identified as G. stordalii was collected at the type locality of G. saxicola in 2002, 2003 (J. Holec) and 2014 (M. Krˇ´ızˇ). The species was also reported from other sites of the Bohemian Switzerland National Park (Holec and Wild 2011). A very similar fungus was found in the Boubı´nsky´ prales virgin forest (Holec et al. 2015) and several other localities in the Czech Republic. All these collections resemble G. saxicola in almost all characters but differ by presence of clamps on basidia. These facts stimulated us to study G. saxicola and G. stordalii in detail using both recent and type material and morphological as well as molecular methods. We ask the following questions: 1. What is the taxonomic status and phylogenetic position of G. saxicola? 2. Is the Czech material preliminary identified as G. stordalii conspecific with the original concept of that species? 3. What is the relation between G. saxicola and G. stordalii?

Materials and methods Morphological study Descriptions of macromorphological characters are based on fresh material collected/photographed by us and cited

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below; those of the holotypes and paratypes are based on published data. Vouchers of our material are kept in the mycological herbarium of the National Museum, Prague (PRM). In addition, collections from Oslo herbarium (O), ˇ eske´ Budeˇjovice (CB), Masaryk Jihocˇeske´ muzeum in C University in Brno (BRNU) and Vlastiveˇdne´ muzeum a galerie in Cˇeska´ Lı´pa, Czech Republic (abbreviated as CELM) were studied. Microscopic characters were recorded from dried material, rehydrated in a 0.5 % solution of Congo Red in ammonia. Spore colour was recorded according to Ku¨ppers (1999) and evaluated in water, 3 % KOH and Melzer’s reagent. The description of microcharacters of Galerina saxicola holotype was based on 40 randomly selected mature spores and 20 randomly selected basidia, cheilocystidia, pileocystidia and caulocystidia. Descriptions of collections from the Kamenice river canyon and Galerina stordalii from Norway were based on at least 20 randomly selected mature spores and ten randomly selected basidia. If present, ten cheilocystidia, pileocystidia and caulocystidia were measured in each specimen. The size of each microscopic structure is given as the 10 and 90 percentiles of all measurements, whereas the 5 and 95 percentiles are given in brackets. All specimens listed under ‘‘Collections studied’’ were examined microscopically; any deviations were recorded and incorporated into the descriptions of the species. Microscopic characters were drawn using an Olympus BX41 light microscope and drawing attachment, and microphotographs were taken using transmitted light or Nomarski contrast, respectively (Olympus BX61 ? DP73 camera). DNA analyses Isolation of DNA from dried specimens followed Holec and Kolarˇ´ık (2013). ITS-LSU rDNA was amplified using primers ITS1F and NL4, and the same primers, together with ITS4 and NL1, were used for sequencing (see Holec and Kolarˇ´ık 2013 for details). Sequences of the ITS and LSU rDNA gene were compared using the nBLAST tool with data deposited in NCBI Genbank (http://blast.ncbi. nlm.nih.gov/Blast.cgi) and the UNITE database (https:// unite.ut.ee). ITS rDNA sequences were combined with related sequences published by Gulden et al. (2005), Curti et al. (2013) and with the most similar sequences deposited in NCBI Genbank and the UNITE database (Fig. 1). Sequences were aligned using MAFFT 6 (Katoh and Toh 2008). The final dataset comprised a total of 53 sequences (of which nine were obtained in this study) and 676 positions, of which 296 were variable and 93 were singletons. Bayesian phylogenetic analyses were carried out in MrBayes 3.0 (Ronquist and Huelsenbeck 2003) software using the same methods as described in Holec and Kolarˇ´ık (2013). The evolutionary model, HKY ? G, was estimated

Galerina saxicola and G. stordalii

Fig. 1 Phylogenetic placement of Galerina saxicola, G. stordalii and G. pruinatipes inferred from ITS rDNA data using Bayesian analysis. The tree is rooted with Flammula alnicola. Sequences printed in bold

were obtained in this study. Statistical support for the branches with Bayesian posterior probability (PP) C 0.5 is shown. The branches with PP C 0.95 are thickened

using MEGA 6.06 (Tamura et al. 2013). Flammula alnicola (Fr.) P. Kumm., member of the sister family Strophariaceae (Matheny et al. 2015), was selected for the outgroup. In addition, we sequenced collections of Galerina pruinatipes A.H.Sm. published by Holec et al. (2015) to enrich our phylogenetic tree.

Results Phylogeny All seven analysed specimens preliminary identified as G. stordalii or G. saxicola had the same ITS-LSU rDNA

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J. Holec et al. Table 1 Collections used for DNA analysis and sequences obtained from NCBI Genbank for comparison Species

Country

Locality

Voucher

Coll. date

DNA sequence (publication)

Galerina saxicola

Czech Rep.

Kamenice river canyon

PRM 685242, HOLOTYPE

1969

Not obtained

Galerina saxicola

Czech Rep.

Kamenice river canyon

PRM 935271a

2014

LT577695 (this paper)

Galerina saxicola

Czech Rep.

Kamenice river canyon

PRM 935272a

2014

LT577693 (this paper)

Galerina saxicola

Czech Rep.

Kamenice river canyon

PRM 896288, EPITYPE

2002

LT577691 (this paper)

Galerina stordalii

Czech Rep.

Galerina stordalii

Czech Rep.

Galerina stordalii

Czech Rep.

Vlcˇ´ı potok stream valley Boubı´nsky´ virgin forest Boubı´nsky´ virgin forest

PRM 923762

2013

LT577692 (this paper)

Galerina stordalii

Czech Rep.

Boubı´nsky´ virgin forest

PRM 923763

2013

LT577694 (this paper)

Galerina stordalii

Norway

Høyfjellsøkologisk forskningsstasjon

O 154169

1979

AJ585435 (Gulden et al. 2005)

PRM 896295

2002

LT577690 (this paper)

PRM 922823

2013

LT577696 (this paper)

Galerina stordalii

Norway

Hellemobotn

O 154179

1983

AJ585434 (Gulden et al. 2005)

Galerina stordalii

Norway

OS401

1998

KC842392 (Stensrud et al. 2014)

Galerina pruinatipes

Czech Rep.

PRM 923041d

2013

LT577697 (this paper)

Galerina pruinatipes

Czech Rep.

Fokkstumyra Boubı´nsky´ virgin forest Boubı´nsky´ virgin forest

PRM 923094d

2013

LT577698 (this paper)

Galerina pruinatipes

France

Chartreusec

O 73438

1979c

AJ585510, AJ871531 (Gulden et al. 2005)

Galerina pruinatipes

USA

Green Lake

MICH 29836, ‘‘TOPOTYPE’’b

1952

AJ585509 (Gulden et al. 2005)

Sequences printed in bold were obtained during this study. For details on vouchers see Collections studied a

Deposited as G. stordalii in NCBI Genbank The collection MICH 29836 = Smith no. 41160 (see http://mycoportal.org/portal/collections/list.php) is not a holotype as indicated by Gulden et al. (2005) but a ‘‘topotype’’collected at the type locality 2 days after the holotype (MICH 10655 = Smith no. 41029) and not cited in the protologue of G. pruinatipes (Mycologia 45: 912, 1953)

b

c

Not given in the cited literature, but kindly provided by the reviewer of this paper

d

Published in Holec et al. (2015: 177, 196)

sequence. Unfortunately, we were not able to obtain sequences from the holotype of G. saxicola in spite of repeated attempts, probably due to the poor state of the material (see notes on G. saxicola below). All three G. stordalii sequences deposited in NCBI GenBank (Table 1) had one to two heterozygotic positions, where one of the base variant also occurred in our sequences, and can thus be considered as 100 % identical to the Czech material (Fig. 1). The most similar taxa were Galerina graminea (Velen.) Ku¨hner (92 % similarity, UNITE code UDB021091, sequence locked) and Galerina clavata (Velen.) Ku¨hner (92 %, AJ585436), both belonging to the Tubariopsis clade, as was shown by Gulden et al. (2005; in that work, G. graminea is included under Galerina laevis Singer). Taxonomic treatment Galerina saxicola Svrcˇek, Zeitschrift fu¨r Mykologie 60(1): 91, 1994. Figs. 2a–e, g, 3a, b. —HOLOTYPE: Czech Republic, Northern Bohemia, Labske´ pı´skovce region, Mezna´ near Hrˇensko, valley of Kamenice river called

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Divoka´ souteˇska, on shaded, mossy, steep, moist sandstone rocks, 120–130 m a. s. l., 30 Jun 1969, M. Svrcˇek (PRM 685242!, Figs. 2a–c, 3a). —EPITYPE (designated here): Czech Republic, Kamenice river canyon between Ticha´ (Edmundova) and Divoka´ souteˇska, 4 Jun 2002, J. Holec JH 34/2002 (PRM 896288!; Figs. 2d, 3b). (Figs. 2a–e, g, 3a, b). Holotype macrocharacters (translation of the original German description by Svrcˇek 1994): Pileus 3–9 mm broad, campanulate, sometimes slightly umbonate, later more convex to depressed, very thin, honey brown to ochre-yellow when fresh, translucently striate, not viscid, pale ochre when dry, light, without distinct veil also when young. Lamellae broad, ventricose, sparse (L = 10–12, l = 1.2), emarginate, pale brown-yellowish, edge finely white floccose. Stipe 10–15 9 1–1.5 mm, completely white, finely pruinose to very finely hairy, not fibrillose. Context without smell, mild, taste not farinaceous. Spore deposit rusty yellow. Holotype microcharacters (based on holotype revision): Spores (8) 8.5–11 9 4–5.5 lm, average 9.7 9 4.7 lm (n = 40), variable, narrowly ellipsoid to fusiform, amygdaliform, sometimes slightly constricted in the middle,

Galerina saxicola and G. stordalii

Fig. 2 Galerina stordalii (= G. saxicola), microcharacters and basidiomata of studied specimens. a Basidia with clamp connections, b spores, c cheilocystidia; G. saxicola, holotype, PRM 685242. d spores; G. saxicola, epitype, PRM 896288. e young basidiomata on mossy sandstone rock; G. saxicola, PRM 935271. f Young

basidiomata on mossy decaying conifer trunk; G. stordalii, PRM 922823. g mature basidiomata on mossy sandstone rock; G. saxicola, PRM 935272. Photographs by M. Vasˇutova´ (a–d), M. Krˇ´ızˇ (e, g), J. Holec (f)

Q = (1.6) 1.7–2.4 (2.6), Qav = 2.0, with distinct apiculus and narrow, indistinct germ pore, slightly verruculose (ornaments very narrow and fine), without plage, ochreyellow in water (S00Y40M10/S00Y70M10), yellowish in KOH (S00Y60M10), light yellow in Melzer’s reagent (S00Y50M00). Basidia (20.5) 22–27 (29) 9 7–8 lm, mostly 4-spored, often with clamps. Cheilocystidia densely packed at the edge, tibiiform, often (ca 30 %) branched,

(27) 28–32 (34) 9 6–9.5 (10) lm, neck 1.5–2.5 lm, capitulum 3.5–4.5 lm, rarely without capitulum. Pileipellis composed of 5–20 lm broad elements, some of them finely incrusted. Pileocystidia numerous, of a similar type as cheilocystidia, but larger and more irregularly shaped, half of them branched up to three times, often growing directly from pileipellis elements, (31) 34–54 (60) 9 (3.5) 4–8 lm, with necks 2–3 (3.5) lm and capitulum (3) 3.5–6 lm, some

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Fig. 3 Galerina stordalii (= G. saxicola), microcharacters of studied specimens: a G. saxicola, holotype, PRM 685242. b G. saxicola, epitype, PRM 896288. c G. stordalii, O 154169. d G. stordalii, PRM

923763. Sp–spores, ba–basidia, che–cheilocystidia, pi–pileocystidia, cau–caulocystidia, bar 10 lm. Drawings by M. Vasˇutova´

of them with yellow pigment. Caulocystidia in dense clusters, mostly of similar shape to cheilocystidia, ca 20 % not capitate, ca 15 % branched, often growing directly from hyphae, (24) 30–50 (55) 9 (5.5) 6–8 (9) lm, neck 2.5–4 lm, capitulum 5–7 (8) lm. Notes: Our study of holotype microcharacters basically agrees with the original description (Svrcˇek 1994). However, the spores appeared more amygdaliform and branched cheilocystidia were discovered. In contradiction with Svrcˇek, we frequently observed clamps at the base of

basidia (Fig. 2a). Unfortunately, the holotype of G. saxicola is in poor condition as it was probably partially rotten (left too long) before drying. Most of the collection is sterile, spores are missing or rare, and some basidia and cheilocystidia are collapsed. We observed a considerable variability of spores, probably caused by non-optimal conditions for spores maturation after picking and drying of the basidiomata. In fertile parts, the spores look ‘‘normal’’, while in almost infertile lamellae regions many are prolonged to fusiform. In all studied regions (15 fragments

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Galerina saxicola and G. stordalii

from various lamellae), clamps are present at the base of basidia only and cheilocystidia are of the same type. This means that the holotype material is homogeneous and represents one species. Recent collections from the type locality: Macrocharacters: Pileus 5–12 mm broad, hemispherical to campanulate when young, then broadly convex-conical, sometimes with a small umbo, thin, hygrophanous, translucently striate almost up to the centre, with darker centre and paler margin, (cream) whitish to pale ochre when young, later pale ochre, yellow-ochre to brown-ochre, sometimes with a fine orange tinge, very finely pruinose at centre (lens), veil absent. Lamellae sparse, L = 12–14, l = 1–3, ventricose, adnexed to emarginate, whitish, then darker, colour the same as pileus, edge concolorous when young, then whitish and finely floccose (lens). Stipe 10–20 9 1–1.5 mm, cylindrical with (sub)bulbous base up to 2 mm broad, slightly uneven, glassy semitranslucent and slightly lustrous, very pale, whitish, later with a yellowish to pale ochre tinge, entirely white pruinose. Context without smell, taste not checked. Spore deposit rusty brown. Microcharacters: Spores 8.5–10 (10.5) 9 4.5–6 lm, average 9.3 9 5.1 (n = 60) amygdaliform, rarely narrowly amygdaliform to ellipsoid, rarely slightly constricted in the middle, Q = (1.5) 1.6–2.1 (2.2), Qav = 1.8, with distinct apiculus and narrow, indistinct germ pore, slightly verrucose to verruculose (ornamentation is narrow, fine to rather prominent), without plage, yellowish in water (S00Y50M10/S10Y40M10), yellowish in KOH (S00Y50M10/S10Y40-50M10), ochre in Melzer’s reagent (S10Y50-60M10/S20Y40M10). Basidia (20) 22–29 (30) 9 (6.5) 7–10 lm, mostly 4-spored, often with clamps. Cheilocystidia densely packed at the edge, tibiiform, rarely or not branched, (20) 24–42 (47) 9 (5.5) 6–11 (12) lm, neck 1.5–3 lm, capitulum (2.5) 3–6 lm. Pileipellis composed of 5–18 lm broad elements, some of them finely incrusted. Numerous pileocystidia of similar type as cheilocystidia, but larger and more irregularly shaped, rarely branched, often growing directly from pileipellis elements, (24) 25–58 (67) 9 (2.5) 4–6 (6.5) lm, with necks 1.5–3 (3.5) lm and capitulum (3) 4–6 (6.5) lm. Caulocystidia growing in dense clusters, mostly of similar shape to cheilocystidia but more variable, very rarely branched, often growing directly from hyphae, (25) 28–57 9 4–11 (12) lm, neck 2–4 (4.5) lm, capitulum 4–9.5 (10.5) lm. Notes: Owing to the poor state of the G. saxicola holotype and the impossibility to obtain its DNA data, we decided to support the taxonomic position of G. saxicola by designating an epitype. The specimen selected as epitype (Figs. 2d, 3b) best fits the holotype in spore size of all

vouchers collected at the type locality. Moreover, there was no problem in characterising its DNA barcode (Table 1). Additional specimens examined: Czech Republic: Northern Bohemia, Labske´ pı´skovce region (currently protected as the Cˇeske´ Sˇvy´carsko National Park), Mezna´ near Hrˇensko, valley of Kamenice river called Divoka´ souteˇska, on shaded, mossy, steep, moist sandstone rocks, 120–130 m a. s. l., 30 Jun 1969, M. Svrcˇek (PRM 685242: holotype).—Ibid., 160 m a. s. l., among mosses on moist sandstone rock, 30 May 2014, M. Krˇ´ıˇz MKD73058 (PRM 935272).—Ibid., adjacent site called Meznı´ mu˚stek, 160 m a. s. l., among mosses on moist sandstone rock, 30 May 2014, M. Krˇ´ıˇz MKD73012 (PRM 935271).—Ibid., Kamenice river canyon between sites called Ticha´ = Edmundova and Divoka´ souteˇska, natural forest: Picea abies, Fagus, Acer pseudoplatanus, etc., 160 m a. s. l., among mosses on moist sandstone rock, 4 Jun 2002, J. Holec JH 34/2002, JH 31/2002, JH 32/2002 (PRM 896288: epitype, 896285, 896286).—Ibid., deep gorge between Soorgrund gorge and Kamenice river canyon, yellow-marked tourist path, natural forest: Picea, Acer pseudoplatanus, Fagus, 190 m a. s. l., among mosses on moist sandstone rock, 15 Jul 2003, J. Holec JH 19/2003 (PRM 915490). Galerina stordalii A.H.Sm. in Smith & Singer, A monograph on the genus Galerina Earle: 203, 1964. Figs. 2f, 3c, d Macrocharacters (translation of the original Latin description by Smith which was based on dried basidiomata and thus lacked some characters): Pileus about 10 mm broad, dirty brown. Lamellae subfulvous, broad, rather distant. Stipe 10–20 mm long, about 1 mm wide, pruinose in upper part, glabrous in lower part. Additional characters mentioned in the protologue: dried pilei 5–15 mm broad, cinnamon brown, no veil remnants evident but there is an evidence of pruinosity, paratypes with pilei 5–10 mm broad, conic, glabrous, striate, probably ochraceous tawny when fresh, stipe not darkening below, ±1 mm diameter. Due to the brevity of the original description, we add data from more detailed descriptions of later collected Norwegian specimens by Gulden (1980, 2012): pileus 3–12 mm, convex to broadly conic to subumbonate, margin straight, becoming reflexed, translucently striate nearly to the centre, shiny, hygrophanous, moist clear yellow to fulvous, paler at margin, lamellae adnexed to adnate, moderately close to subdistant, pale yellow to warm ochre, edge minutely fimbriate, stipe 12–30 9 1–1.5 mm, equal, fistulose, minutely pruinose all over, first whitish, but slightly yellow to reddish brown with age. Microcharacters (based on our revision of Norwegian collections O 154169 and O 154179): Spores 8.5–10 9 5–6 lm, average 9.2 9 5.2 (n = 40), amygdal-

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iform, sometimes narrowly ellipsoid, Q = (1.5) 1.6–1.9, Qav = 1.8, with distinct apiculus and narrow, indistinct germ pore, slightly verruculose to verrucose (ornamentation narrow, fine to rather prominent), without plage, yellowish in water (S00-10Y50M00-10), yellowish in KOH (S00-10Y50M00-10), light ochre in Melzer’s reagent (S1020Y60M10-30-Y70M50C20). Basidia 22–33 9 7–11 lm, mostly 4-spored, often with clamps. Cheilocystidia densely packed at the edge, tibiiform, rarely to frequently branched (10–30 % per collection), 24–35 9 5–9 lm, neck 2–3 lm, capitulum 4–6 lm, rarely without capitulum. Pileipellis not studied because of small amount of material. Caulocystidia (specimen O 154169) growing in dense clusters, mostly of similar shape as cheilocystidia, rarely non-capitate, often branched, often growing directly from hyphae, 42–62 9 11–13 lm, neck 2.5–4 lm, capitulum 7–8 lm. Notes: Due to the age of the G. stordalii type material, collected in 1947, we refrained from attempts to isolate its DNA. For comparison with G. saxicola (see Discussion), we used more recent collections of G. stordalii from its type country—Norway, identified and sequenced by G. Gulden, a renowned Galerina specialist. Additional specimens examined: Norway: Hordaland, Ulvik, Finse, at Høyfjellsøkologisk forskningsstasjon, in Sphagnum, Empetrum, Polytrichum, 8 Aug 1979, G. Gulden (O 154169, GenBank AJ585435).—Nordland, Tysfjord, Hellemobotn, on Sphagnum with Polytrichum, 26 July 1983, G. Gulden (O 154179, GenBank AJ585434). ˇ eske´ Sˇvy´carsko National Park, Czech Republic: C 5.5 km W of Kra´sna´ Lı´pa, Kyjovske´ u´dolı´ valley: Vlcˇ´ı potok stream valley near Turisticky´ most (N side), manmade spruce forest on sandstone bedrock, in places with Alnus incana, 330 m a. s. l., among mosses on moist sandstone rock, 6 Jun 2002, J. Holec JH 47/2002 (PRM 896295).—Cˇeska´ Lı´pa District, NE of Doksy, BrˇehyneˇPecopala National Nature Reserve, bog spruce forest in NE part of Brˇehynˇsky´ rybnı´k pond, ca 1.9 km E of Mly´nsky´ vrch hill, 7 Jun 2012, A. Lepsˇova´ and L. Zı´barova´ (CB).— Jizerske´ hory Mts., NW of Jizerka, Rasˇelinisˇteˇ Jizerky National Nature Reserve, 50°500 00.900 N, 15°200 00.700 E, bog spruce forest, 877 m a. s. l., in Polytrichum, 21 July 2013, J. Slavı´cˇek (CELM 10463).—Ibid., 215 m NNE of ‘‘Hnojovy´’’ house, 50°490 35.900 N, 15°200 17.600 E, roadside at margin of spruce forest, 869 m a. s. l., 26 Jun 2012, J. Slavı´cˇek (CELM 10399).—Sˇumava Mts., Za´tonˇ near Lenora, Boubı´nsky´ prales virgin forest, core area, E and ESE slopes, natural montane forest (Fagus, Picea, Abies), 950 m a. s. l., in moss on decaying trunk of Picea abies, 12 Jun 2013, J. Holec JH 45/2013 (PRM 922823).—Ibid., in moss on decayed trunk, 12 Jun 2013, M. Krˇ´ıˇz (PRM 923762).—Ibid., E and ENE slopes above core area, ±1070 m a. s. l., in moss on decayed trunk of a conifer (Picea? Abies?), 27 Jun 2013, M. Krˇ´ıˇz (PRM 923763).—

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Jesenı´ky Mts., W of Karlova Studa´nka, Pradeˇd National Nature Reserve, near Bı´la´ Opava waterfall, in mosses on soil in mountain spruce forest, 1100–1150 m a. s. l., 1 Aug 2010, D. Dvorˇa´k (BRNU 634651).

Discussion Galerina saxicola—type locality, variability, related species Svrcˇek (1994) based its description of Galerina saxicola on only one collection (holotype) although he mentioned that the species is rather common at the type locality. His description of macrocharacters fully agrees with recent material from the type locality. Similar to Svrcˇek, we observed that G. saxicola is common at the type locality. The site is very distinct as it represents a deep river canyon delimited by vertical sandstone rocks and is under the influence of climatic inversion. Even if situated at a low altitude, its microclimate is cold and humid which enables an unusual (extrazonal) occurrence of some boreal-montane fungi, e.g. Phellinus nigrolimitatus (Holec 2009). The rock walls are usually covered by mosses and liverworts. This is the typical habitat of G. saxicola. Other Galerina species growing there clearly differ by their macrocharacters. G. saxicola is characteristic by its whitish, glassy, pruinose, slightly uneven stipe and rather pale, translucently striate pileus with sparse lamellae. Even if it is a small fungus, its habit and consistency is rather ‘‘robust’’ as the stipe is relatively thick in relation to its length and cannot easily be broken. Concerning microcharacters, the recently collected material described in Results is almost identical to the holotype of G. saxicola. The only discrepancies are the presence of extremely narrow spores in the holotype (however, they are rarely present in the recent material, too), minor differences in the proportion of branched cheilocystidia and caulocystidia lacking a capitulum. The latter characters are variable and differ between collections from the same locality. In our opinion, these discrepancies fall under the general variability of G. saxicola. Svrcˇek (1994) wrote that G. saxicola is closest to G. dimorphocystis A.H.Sm. & Singer and G. propinqua Bas. He also commented on G. stordalii. We agree with his selection of the most similar species. Galerina propinqua, described from Germany growing on bare and mossy peat and on Sphagnum (Bas 1965), has already been synonymised with G. stordalii by Gulden (1980). The identity of G. dimorphocystis remains unclear. It was described from ‘‘moss, especially over mossy logs in bogs or wet hillsides’’ in North America (Smith and Singer 1955) as a species lacking clamps and possessing large vesiculose

Galerina saxicola and G. stordalii

cheilocystidia near the cap margin. Unfortunately, its holotype is probably missing (Gulden and Vesterholt 1999: 691) and paratype material studied by Gulden (1980) possesses clamps and spores with a pore. Ku¨hner (1972) reported G. dimorphocystis from Scandinavia but interpreted it as a fungus with clamps on the basidia and partly also on the pileocystidia. Galerina dimorphocystis sensu Ku¨hner was synonymised with G. stordalii by Gulden and Vesterholt (1999: 690). Galerina stordalii A.H.Sm. is a species based on Norwegian collections from ‘‘Sphagnum and other mosses’’ (Smith in Smith and Singer 1964: 204) distinguished by the broadly conical cap, the ventricose, subdistant gills, the nearly white, entirely pruinose stipe, the small spores with apical pore and the presence of clamps, best seen in young basidia (Gulden et al. 1985; Gulden and Vesterholt 1999; Gulden 2012). According to Svrcˇek (1994), G. saxicola differs from G. stordalii by a total absence of clamps, growth on foliose liverworts and saxicolous mosses (not on Sphagnum), and spore shape. However, our revision of G. saxicola showed that it does possess clamps and most of its characters, including those mentioned as diagnostic by Svrcˇek (1994), agree with G. stordalii (Table 2; Figs. 2, 3). The conspecificity of G. saxicola and G. stordalii is supported by molecular data (Fig. 1), too. Galerina stordalii—taxonomy, ecology, distribution Almost all characters of Czech collections preliminary identified as G. stordalii agree with the original description of G. stordalii (Smith in Smith and Singer 1964), later published descriptions (Gulden 1980, 2012; Gulden et al. 1985; Gulden and Vesterholt 1999) and the Norwegian collections studied by us. Moreover, the ITS DNA sequence of the Czech collections is identical to the Norwegian ones (Fig. 1). The differences concern clamps and ecology. We observed clamps only at the base of the basidia (frequently and regularly), whereas some authors report them in other tissues, too: ‘‘also scattered on hymenial hyphae and on hyphae of cortical layers’’ (Gulden and Vesterholt 1999), ‘‘scattered in subhymenium’’ (Gulden et al. 1985), ‘‘at some septa’’ (Gulden 2012). The discrepancies could mean that clamp frequency and occurrence vary between collections, which is a well-known fact in some genera of agarics. Concerning ecology, Gulden and Vesterholt (1999) write that G. stordalii ‘‘grows on peat and moss in ombrotrophic bogs and apparently is confined to Sphagnum’’. Later, Gulden (2012) characterised it as a fungus of ‘‘bogs, peat and moist snow-beds, mostly on Sphagnum’’. However, in the Norwegian collections studied by us, Polytrichum was present in all cases. Moreover, ‘‘Sphagnum and other mosses’’ are mentioned in the original

description (Smith in Smith and Singer 1964: 204). The ecology of Czech records is more variable: one is from a Sphagnum-Polytrichum stand in a boggy spruce forest at a bog margin, one from a moist sandstone rock, another two from a mossy roadside and the other ones from unidentified mosses (not Sphagnum) on decaying fallen trunks of conifers in old-growth montane forests (Boubı´nsky´ prales: see Holec et al. 2015, Pradeˇd). The occurrence in old-growth forests agrees with data from Poland (Wojewoda 2003: Białowie_za virgin forest; Ronikier 2012: Sarnia Skała massif, records from coniferous wood covered with mosses). It seems that the studied fungus is a non-specialised saprotroph decaying dead parts of various mosses. In this situation, when all other characters (including molecular ones) agree with those of G. stordalii, we consider the Czech material conspecific with the Norwegian one. The differences in iodine reaction (Svrcˇek 1994: brownish red to wine red, i.e. distinctly dextrinoid; Gulden and Vesterholt 1999; Gulden 2012: faintly dextrinoid) can be attributed to possible differences in iodine concentration in Melzer’s reagent used and subjectivity of the observation. Our collections also differed in the frequency of branched cheilocystidia. We did not find any correlating diagnostic character and consider this fact a common intraspecific variability. Smith and Singer (1964) report frequent occurrence of mucilaginous cheilocystidia heads or slightly thickened cheilocystidia wall. This character was not observed by us or other authors. De Haan and Walleyn (2009) describe an unusually long stipe in G. stordalii (25–45 mm). The fact that fungi growing in deep moss sometimes have a very long stipe is generally known. However, the authors give slightly shorter spores ((7.6) 8–9 (9.8) lm) and use the term ‘‘hairs’’ for pileus and stipe coverage which is in contradiction to our observations and data of all other authors, describing the surface as minutely pruinose. These facts suggest that records by De Haan and Walleyn (2009) could differ slightly from the typical G. stordalii. Distribution: Galerina stordalii is well documented from North and Central Europe: Norway including Svalbard, Finland, Faroe Islands, Iceland (e.g. Gulden 2012), Sweden (Ku¨hner 1972 as G. dimorphocystis, Gulden 2012), The Netherlands (Gulden et al. 1985, http://www.ver spreidingsatlas.nl), Germany (e.g. Bas 1965, as G. propinqua), Czech Republic (this paper), Austria (Austrian Mycological Society 2009) and Poland (Wojewoda 2003; Ronikier 2012). The species is also found in Scotland (http://www.fieldmycology.net), and there are internet data from France (revision needed). It is further known from Greenland (Gulden 2006). As shown above, the slightly differing records from Belgium (De Haan and Walleyn 2009) should be revised. Gulden (1980) cited collections from North America by synonymisation of records

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J. Holec et al. Table 2 Comparison of spore characters of Galerina saxicola and G. stordalii G. saxicola holotype!

G. saxicola epitype!

G. stordalii type material, data from Smith and Singer (1964)

G. stordalii recent material from Norway, see collections studied

G. stordalii data from Gulden (2012)

Spore size

(8) 8.5–11 9 4–5.5

8.5–10 (10.5) 9 (4) 4.5–5.5

9–11 9 5–5.5

8.5–10 9 5–6

(7) 9–11 9 4.5–6 (6.5)

Average spore size Spore shape

9.7 9 4.7 (n = 40)

9.6 9 4.8 (n = 20)

Narrowly ellipsoid to fusiform, amygdaliform, sometimes slightly constricted in the middle

Amygdaliform to narrowly amygdaliform, narrowly ellipsoid

Q

(1.6) 1.7–2.4 (2.6), Qav = 2

1.8–2.2 (2.5), Qav = 2

published as G. dimorphocystis and G. frigida. There is also an Asian record from Siberia (Nezdojminogo 1985) under the name of G. dimorphocystis (Gulden and Vesterholt 1999: 690). Galerina stordalii is included in the Czech Red Book (Kotlaba et al. 1995: 102–103), based on a record or records from alpine bogs in the Krkonosˇe Mts. (Giant Mts., Riesengebirge). This information is hard to verify taxonomically due to the absence of any data on publicly accessible vouchers.

Conclusions Collections of G. stordalii from the Czech Republic are clearly conspecific with the Norwegian ones, represented by vouchers from the type country, identified and sequenced by G. Gulden, renowned Galerina monographer. Galerina saxicola proved to be conspecific with G. stordalii and being a later published name, it has to be considered synonym of G. stordalii. The full synonymy of G. stordalii is summarised below, where also its diagnostic characters and newly amended data on ecology and distribution are mentioned. In addition, corrected data on type collections of G. stordalii are provided based on information from the anonymous reviewer of this paper and the literature. Galerina stordalii A.H.Sm. in Smith & Singer, A monograph on the genus Galerina Earle: 203, 1964.— HOLOTYPE: Norway, Hedmark County, municipality of Trysil, near Odden on the W side of Ossjøen, ca. 450 m a. s. l., 3 Jun 1947, Lars Odden s.n. (coll. J. Stordal no. 376, MICH [n.v.]). —ISOTYPE: O [n.v.], (see Gulden 1980: 229, incorrectly labelled as holotype).—PARATYPES: Norway, ‘‘Blandt mose Hedmark: Trysil hd., Odden Lars’’,

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9.2 9 5.2 (n = 40)

Inequilateral in profile, narrowly ovate in face view, often obscurely angular

Amygdaliform, sometimes narrowly ellipsoid

Amygdaloid, ±angular, with acute apex

(1.5) 1.6–1.9, Qav = 1.8

on Sphagnum and other mosses, J. Stordal nos. 1177, 1792 (O [n.v.]). = Galerina propinqua Bas, Persoonia 3: 360, 1965 (based on revision by Gulden 1980). = Galerina frigida V.L.Wells & Kempton, Lloydia 32: 375, 1969 (based on revision by Gulden 1980). = Galerina saxicola Svrcˇek, syn. nov., Zeitschrift fu¨r Mykologie 60(1): 91, 1994 (revision published here). Misapplied name for Galerina stordalii: Galerina dimorphocystis A.H.Sm. & Singer sensu Ku¨hner 1972; Nezdojminogo 1985 (based on revisions by Gulden 1980, Gulden and Vesterholt 1999). Notes: According to the reviewer of this paper, Smith’s data on types should be corrected as follows: Lars Odden is the name of the collector, J. Stordal’s collection 1177 is not from Fordsland (Smith and Singer 1964: 204), which is not even a place in Norway, but from Bavalen in the municipality of Voss in the county of Hordaland, Norway. According to Gulden (1980), collection 1177 is a mix of three species: G. clavata, G. vittiformis, G. stylifera. Diagnostic characters (based on collections studied by us): Small fungus, pileus 5–15 mm, broadly convex-conical when mature, pale ochre, yellow-ochre to brown-ochre with paler margin, hygrophanous, translucently striate almost up to the centre, lamellae sparse, distant, ventricose, with whitish, finely floccose (denticulate) edge, stipe 10–30 9 1–1.5 mm, slightly uneven, pale, whitish, later with a yellowish to pale ochre tinge, glassy semitranslucent, entirely white pruinose, spores (8) 8.5–10.5 (11) 9 (4) 4.5–6 lm, Qav = 1.8–2, variable in shape, typically amygdaliform but also narrowly ellipsoid to fusiform, sometimes slightly constricted in the middle and/ or with a conically acute apex (the spores then looking ‘‘angular’’), verruculose, without plage, with narrow germ

Galerina saxicola and G. stordalii

pore, basidia mostly 4-spored, often with clamp at base, cheilocystidia tibiiform, often branched, pleurocystidia absent, pileo- and caulocystidia resembling cheilocystidia but larger and more irregularly shaped. Clamps absent except for basidia (or very rare in other tissues according to the literature cited in Notes). Distribution and ecology: Rare in arctic, alpine, boreal and temperate regions of Europe, Asia and North America, probably a saprotroph growing on Sphagnum and other mosses or peat, in bogs, moist snow-beds, boggy spruce forests, on decaying conifer trunks in old-growth forests and on moist sandstone rocks in a river canyon under the influence of climatic inversion. Acknowledgments We thank our colleagues Lucie Zı´barova´, Daniel Dvorˇa´k and Josef Slavı´cˇek (Czech Republic) for providing their specimens of Galerina stordalii, Gro Gulden (Norway) for help with the literature, and staff of Oslo herbarium for arranging a loan of G. stordalii collections. The anonymous reviewer of this paper is warmly acknowledged for his valuable comments. The work by J. Holec and M. Krˇ´ızˇ was financially supported by the Ministry of Culture of the Czech Republic (DKRVO 2016/08, National Museum, 00023272).

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