Introduction

Hodophilus foetens (W. Phillips) Birkebak and Adamčík is a gilled member of the family Clavariaceae Chevall. (Birkebak et al. 2016). It had been previously included within the genera Hygrophorus Fr. (Dennis 1953; Singer 1959; Hesler and Smith 1963) and Camarophyllopsis Herink (Boertmann 2012; Kovalenko et al. 2012). The species is the type of the genus Hodophilus R. Heim ex R. Heim that is characterized by a hymeniderm pileipellis composed of globose, obpyriform to sphaeropedunculate terminal elements and absence of clamp connections (Birkebak et al. 2016). It is traditionally recognised as the sole European species with an unpleasant strong odour (Boertmann 2012) variously interpreted as similar to that of Tricholoma sulphureum (Bull.) P. Kumm. or Thelephora palmata (Scop.) Fr. (e.g. Phillips 1878; Heim 1969; Moser 1978): strong, fetid, gas-like (Boertmann 2012) or naphthalene-like (Courtecuisse and Duhem 1994). This odour is the main distinguishing character for the species in Europe. For the odour terminology we follow Ghyselinck (2003) and Adamčík et al. (2016) who analysed odour descriptions of Ho. foetens by various authors and concluded that the best fitting term to describe it is naphthalene-like (similar to the odour of mothballs). Recently, Kovalenko et al. (2012) recognised an additional European species with a naphthalene odour, Camarophyllopsis albofloccipes A.E. Kovalenko, E.F. Malysheva and O.V. Morozova, based on material from Russia.

In contrast to the situation in Europe, four Hodophilus taxa (treated in the genus Hygrophorus) with strong odours are recognised from North America (Hesler and Smith 1963), including their interpretation of the European type species. Our recent study using a multi-locus phylogeny combined with new morphological data (Adamčík et al. 2016) recognised five North American Hodophilus taxa with a naphthalene odour, two described as new. European collections with this odour are clustered in four different well-supported clades, probably representing different phylogenetic species.

In this study, we focus on European members of Hodophilus with a naphthalene odour. We aim to recognise how many phylogenetic species occur in the studied European sampling, their morphological distinguishing characters and which of the species clades correspond to the original concept of Ho. foetens.

Materials and methods

Taxon sampling

For the backbone of our study, we used sampling and sequence data published in Adamčík et al. (2016). The sequence dataset of that study comprises, altogether, 34 Hodophilus collections with a naphthalene odour: 14 North American and 20 European. We supplemented this with an additional 12 European collections of Hodophilus with a naphthalene odour, including the type of C. albofloccipes, two Hodophilus collections without such odour and one collection identified as C. atrovelutina (Romagn.) Argaud. All samples used in this study are listed in Supplementary Table 1. The concept of Ho. foetens (sensu stricto) is based only on morphological observations because attempted PCR amplifications of DNA extracts from the type collection were unsuccessful (Adamčík et al. 2016).

DNA extraction, PCR, and sequencing

Three gene regions (nrLSU, nrITS and rpb2) were amplified, sequenced and analysed. Protocols of Birkebak et al. (2013) were followed for DNA extraction, PCR, and sequencing. Primer pairs ITS1F-ITS4 (Gardes and Bruns 1993; White et al. 1990) were used to amplify the ITS region. Combinations of LR0R-LR7, LR0R-LR5, or LR0R-LR16 (http://sites.biology.duke.edu/fungi/mycolab/primers.htm) were used to amplify and sequence the nLSU region. The primer pair b6F and b7.1R (Matheny 2005) was used to amplify and sequence the most variable region of the rpb2 gene between conserved domains 6 and 7. Sequencing was performed at the University of Tennessee Genomics Core facility (Knoxville, Tennessee, USA) and at the SEQme sequencing company (Dobříš, Czech Republic).

Phylogenetic analyses

Alignments for individual regions were created in CLUSTAL X (Larkin et al. 2007) and manually adjusted by eye in AliView (Larsson 2014). Individual alignments were concatenated in SeaView version 4 (Gouy et al. 2010). PartitionFinder (Lanfear et al. 2014) was used to identify the best partition scheme and molecular models under the AICc criterion. Maximum likelihood (ML) phylogenetic reconstruction was performed with RAxML version 7.4.2 (Stamatakis et al. 2008) implemented in RAxML GUI (Silvestro and Michalak 2012) with 1000 bootstrap replicates. Bayesian inference (BI) was performed in MrBayes v3.2.2 (Ronquist et al. 2011) running ten million generations and sampling parameter states and trees every ten thousand generations. In order to ensure convergence had been reached, the average standard deviation of split frequencies was monitored to ensure it fell below 0.01, and trace files of the parameters were examined to ensure proper mixing. A 25% burn-in was used. We consider bootstrap values >70% and posterior probabilities >0.95 as strong support for clades. Bootstrap values between 50 and 70% and posterior probabilities between 0.80 and 0.95 can be considered as moderate support for clades. States and provinces for the United States and Canada are abbreviated and country abbreviations follow the three-letter ISO code (International Organization for Standardization, Geneva, Switzerland). All sequences are deposited in GenBank. The concatenated final alignment has been deposited at TreeBASE (19902).

Morphological analyses

Macromorphological descriptions were prepared from fresh material shortly after collection from the field. However, in some cases when such data were limited (because older collections were all identified directly as C. foetens without field descriptions), we also used photographs to supplement the descriptions or to verify some macromorphological characters. The number of full length lamellae was counted (i.e. those spanning the whole gill region) and is treated in the species descriptions as “L”. The number of short lamellulae between each pair of full length lamellae is labelled as “l” (Bas et al. 1988). Lamellae of dry specimens were also observed under a stereo microscope (Zeiss model 437166). Colour nomenclature standards follow Kornerup and Wanscher (1967).

Microscopical structures were examined on desiccated herbarium specimens in Congo red solution with ammonia after a short treatment in aqueous 10% KOH. Features were observed under an Olympus CX-41 light microscope with an oil-immersion lens at a magnification of 1000×. All drawings of microscopic structures, with the exception of basidiospores, were made with a camera lucida using an Olympus U-DA drawing attachment at a projection scale of 2000×. Basidiospores were scanned with an Artray Artcam 300MI camera and measured by Quick Micro Photo (version 2.1) software. Enlarged scanned pictures of spores were used for measuring with an accuracy of 0.1 μm and for making line drawings. Q-value is the length/width ratio of basidiospores. Statistics of microscopic dimensions are based on 30 measurements and given as a mean value plus/minus standard deviation; values in parentheses give measured minimum or maximum values. Basidiospores were tested in Melzer’s reagent for amyloid or dextrinoid reactions (Moser 1978).

Older or insufficiently preserved specimens of Hodophilus specimens display collapsed hyphal structures in the pileipellis. Hyphal terminations often form compact conglomerates that do not dissolve in the KOH solution. This makes it especially difficult to trace septa and can cause a bias in length values and length/width ratio values of the terminal cells. For this reason, such insufficiently conserved specimens were eliminated from our morphological analyses, with exception of the Ho. foetens type. In our analysis of micromorphological characters, we included all characters used in our previous study on North American Hodophilus species with a naphthalene odour (Adamčík et al. 2016). In addition, dimensions of subterminal cells of hyphae in the pileipellis are used because we observed that they are frequently inflated, sphaeropedunculate or clavate in the Ho. foetens type. Pileipellis elements near the pileus margin and the pileus centre were observed and evaluated separately.

Results

Phylogenetic analyses

All nodes recovered from the ML inference were also recovered by the BI analysis. The tree topology and grouping of individual collections (Fig. 1) are nearly identical with results of our previous study on North American Hodophilus species with a naphthalene odour (Adamčík et al. 2016). The majority of European collections form four well supported clades that do not merge with any North American collections, and all but two (discussed below) belong to the Ho. foetens superclade. Based on morphology (explained below) one clade is assigned to Ho. foetens, and three clades correspond to new species described in this study: Ho. pallidus Adamčík, Jančovičová & Looney, Ho. subfoetens Adamčík, Jančovičová & Looney and Ho. tenuicystidiatus Jančovičová, Adamčík & Looney. One difference from the previous study is a newly added collection K(M)175255 that represents a singleton sample in the tree. Our phylogenetic reconstruction clearly suggests that it is an autonomous species, but it is not described and recognised morphologically here because sufficient sampling and macromorphological data are not available. The other ten new samples of Hodophilus with a naphthalene odour are clustered in Ho. foetens (six new collections, 13 in total), Ho. tenuicystidiatus (three new collections, ten in total) and Ho. subfoetens (one new collection, five in total). Collections identified as Ho. atropunctus based on dark punctuations on the surface of the stipe and an indistinct odour are partly placed in a strongly supported clade within the Ho. foetens superclade (four collections), but two such collections are placed in the Ho. micaceus superclade and are clustered in one group (H. micaceus group 2) together with collections without dark punctuations.

Fig. 1
figure 1

Maximum Likelihood phylogeny inferred from three loci (ITS, LSU, and rpb2) with species-level clades highlighted as well as the two known superclades composing the genus Hodophilus. The species clade names in green are North American and in black European. Collection labels are updated with appropriate taxon labels except where collector identifications disagree. In blue are new samples added in this study. Also included are collection labels, country (and in some cases state/province), and whether this represents a type collection. Bootstrap values followed by Bayesian posterior probabilities are indicated at nodes

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The type of C. albofloccipes is placed in the genus Hodophilus, and although it is described as having an odour identical to Ho. foetens, it is placed in the H. micaceus superclade in a terminal group labelled as Ho. micaceus clade 3. This strongly supported group contains another four collections, but at least three of them lack any distinctive odour. One collection from Kew [K(M)161018], originally identified as Ho. (Camarophyllopsis) foetens, is placed in the Ho. micaceus superclade within the group Ho. micaceus group 1, but we were not able to ascertain whether this was identified based on the presence of a naphthalene odour.

Morphological delimitation of genetically defined groups

Only a single Hodophilus species with a naphthalene odour had been recognised (Ho. foetens) prior to the discovery of Ho. albofloccipes by Kovalenko et al. (2012), and thus the morphological observations presented here are based mainly on notes describing samples collected after 2013. The only older, fully described collection is SAV F-3488 belonging to Ho. subfoetens, represented only by older collections (2002–2005). Hodophilus pallidus is represented by two collections that were documented by photos in the field, but have no description of macromorphological characters. The macromorphological description of Ho. tenuicystidiatus was prepared based on two recent collections (SLO725 and SLO772) and of Ho. foetens (sensu stricto) is based on five collections (SAV F-4447, SAV F-4448, SLO673, SLO769 and SLO770). The differences in field aspect are summarised and compared in Table 1.

Table 1 Comparison of field characters observed from European Hodophilus taxa with a naphthalene odour according to our observations and for Hodophilus albofloccipes according to Kovalenko et al. (2012)
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Based on Kovalenko et al. (2012), it seems that the recently described C. albofloccipes is different from all other Hodophilus species with a naphthalene odour. It is described as having a yellow coloured pileus and stipe surface, with the latter bearing fine, white granules or scales on the stipe surface. However, the species is probably variable in colour, because in our phylogeny, it is grouped with four other collections and at least two of them (PAM06091103, SAV F-3032) had no distinctive yellow colour or white granulations. Based on our morphological investigation of the type, micromorphological characters offer a more decisive delimitation of the species from other European species with a naphthalene odour. Hodophilus albofloccipes (type) has very numerous small (length and width up to 5 μm) subterminal and subsequent cells in the pileipellis, the character that defines the whole Ho. micaceus superclade (Adamčík et al. 2016). In addition, we confirm the presence of the very specific caulocystidia observed by Kovalenko et al. (2012). The caulocystidia are not only nodulose, but often very twisted and lobate.

The dark brown colour of basidiomata described and illustrated in the protologue of Ho. foetens (Phillips 1878, as Hygrophorus foetens) clearly corresponds to our observations of basidiomata that turn partly black during maturation and drying (Table 1). In agreement with this are micromorphological characters (Table 2, Fig. 2): caulocystidia are the widest among all observed European species, and the subterminal cells of hyphae in the pileipellis are more frequently inflated (especially near the pileus margin). This species was described from Wales, and accordingly, it is the most frequent among studied material from the UK (Fig. 1). The other three species are briefly distinguished here as: Ho. tenuicystidiatus by closer lamellae and very narrow caulocystidia, Ho. subfoetens and Ho. pallidus both with more distant lamellae and differing from each other by shorter terminal cells in the pileipellis near the pileus centre of the latter (Tables 1, and 2).

Table 2 Average values of 30 measurements of selected micromorphological characters observed on studied Hodophilus taxa with an unpleasant odour
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Figs. 2–5
figure 2

Hodophilus foetens (holotypus). 2. Hyphal terminations in pileipellis near the pileus margin. 3. Hyphal terminations in pileipellis near the pileus centre. 4. Caulocystidia. 5. Spores, basidia and basidiola. Scale bar = 10 μm, spores are 50% more magnified

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Taxonomy

Dichotomous key to European Hodophilus species with a napthalene odour

1 Pileus and stipe surface usually with conspicuous yellow tints; subterminal cells in pileipellis frequently small (shorter than 5 μm); caulocystidia often irregularly inflated, nodulose, lobate and twisted

Hodophilus albofloccipes

1* Basidiomata without or with only dull yellow-brownish colours; subterminal cells in pileipellis rarely small; caulocystidia rarely lobate or twisted

2 Lamellae moderately close (L = 18–30)

3 Basidiomata becoming dark brown to black, especially near the pileus margin upon maturation or drying; caulocystidia on average wider than 7.5 μm

Hodophilus foetens

3* Mature or dry basidiomata not becoming darker; caulocystidia on average up to 7 μm wide

Hodophilus tenuicystidiatus

2* Lamellae distant [L = 10–18(–20)]

4 Pileus orange grey to greyish orange when fresh, drying pale orange; terminal cells of hyphae near the pileus centre with ratio of length/width mainly <2.5

Hodophilus pallidus

4* Pileus brown, grey brown, brownish grey, or when dry dark brown; terminal cells of hyphae near the pileus centre with ratio of length/width mainly >2.5

Hodophilus subfoetens

Hodophilus albofloccipes (A.E. Kovalenko, E.F. Malysheva & O.V. Morozova) Looney & Adamčík, comb. nov.

≡ Camarophyllopsis albofloccipes A.E. Kovalenko, E.F. Malysheva & O.V. Morozova, Mikol. Fitopatol. 46: 64. 2012 (Basionym).

MycoBank No.: MB 818293

Hodophilus foetens (W. Phillips) Birkebak & Adamčík, Mycologia 108(5): 866. 2016

Figs. 2–5, 6–7, 13, 17, 21, 25

Figs. 6–12
figure 3

Basidiomata field aspect of 6. Hodophilus foetens (SAV F-4443) photo by Z. Egertová. 7. Hodophilus foetens (SLO770) photo by S Jančovičová. 8. Hodophilus tenuicystidiatus (SLO725, holotypus) photo by S Jančovičová. 9. Hodophilus tenuicystidiatus (PAM14102901) photo by P.-A. Moreau. 10. Hodophilus subfoetens (SAV F-3497) photo by P Marstad. 11. Hodophilus pallidus (SAV F-3486, holotypus) photo by S Jančovičová. 12. Hodophilus pallidus (SAV F-3489) photo by S Jančovičová. Scale bar = 1 cm

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Figs. 13–16
figure 4

Caulocystidia of 13. Hodophilus foetens (SAV F-4448, epitypus). 14. Hodophilus tenuicystidiatus (SAV F-3850). 15. Hodophilus subfoetens (SAV F-3488, holotypus). 16. Hodophilus pallidus (SAV F-3486, holotypus). Scale bar = 10 μm

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Figs. 17–20
figure 5

Spores, basidia and basidiola of 17. Hodophilus foetens (SAV F-4448, epitypus). 18. Hodophilus tenuicystidiatus (SAV F-3850). 19. Hodophilus subfoetens (SAV F-3488, holotypus). 20. Hodophilus pallidus (SAV F-3486, holotypus). Scale bar = 10 μm, spores are 50% more magnified

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Figs. 21–24
figure 6

Hyphal terminations in pileipellis near the pileus margin of 21. Hodophilus foetens (SAV F-4448, epitypus). 22. Hodophilus tenuicystidiatus (SAV F-3850). 23. Hodophilus subfoetens (SAV F-3488, holotypus). 24. Hodophilus pallidus (SAV F-3486, holotypus). Scale bar = 10 μm

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Figs. 25–28
figure 7

Hyphal terminations in pileipellis near the pileus centre of 25. Hodophilus foetens (SAV F-4448, epitypus). 26. Hodophilus tenuicystidiatus (SAV F-3850). 27. Hodophilus subfoetens (SAV F-3488, holotypus). 28. Hodophilus pallidus (SAV F-3486, holotypus). Scale bar = 10 μm

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≡ Hygrophorus foetens W. Phillips, Grevillea 7(42): 74. 1878.

≡ Camarophyllus foetens (W. Phillips) J.E. Lange, Dansk. Bot. Ark. 4(4):18. 1923

≡ Aeruginospora foetens (W. Phillips) M.M. Moser, Röhrlinge-Blätterpilze, 3rd ed.: 69. 1967

≡ Hygrocybe foetens (W. Phillips) P.D. Orton & Watling, Notes Roy. Bot. Gard. Edinburgh 29: 134. 1969

≡ Hygrotrama foetens (W. Phillips) Singer, Beih. Sydowia 7: 3. 1973

≡ Camarophyllopsis foetens (W. Phillips) Arnolds, Mycotaxon 25(2): 643. 1986

MycoBank No.: MB810136

Holotypus: K(M) 92776.

Epitypus (designated here): SAV F-4448.

Diagnosis: Pileus with brown colours that become darker brownish black when mature or dry, especially near the margin; lamellae moderately close (L = 18–26), stipe near lamellae usually with darker fibrils or punctuations, flesh with a strong naphthalene odour; caulocystidia obtuse, mainly longer than 30 μm and wider than 8 μm (26.5–61 × 6.5–11.5 μm), towards bases usually attenuated or pedunculate and flexuous; pileipellis a hymeniderm, near the pileus margin composed of sphaeropedunculate, obpyriform or clavate terminal cells, subterminal cells of hyphae near the pileus margin often inflated and wider than 8 μm.

Pileus 5–20(40) mm broad, convex to plano-convex, sometimes weakly depressed near the centre, rarely with indistinct low umbo; margin inflexed, later straight, crenate, when wet translucently striated at 1/3 to 2/3; surface matt, smooth, but towards the centre slightly rugulose or veined, when dry cracked into irregular, blackish scales; weakly hygrophanous, fresh young basidiomata light brown (clay 5D5), yellowish brown (bronze 5E5) or dark brown (chocolate brown 6F4), towards the margin greyish orange (5B3) or light brown (bronze 6D5), when dry or mature turning at spots or completely brownish black (5F3). Stipe 15–62(70) × 1–4 mm, cylindrical and narrowed towards the base, usually flexuous, sometimes compressed or longitudinally grooved, smooth, shiny, at the apex usually with darker fibrils or punctuations, at the base sometimes white tomentose; often at the apex light brown (blonde 5D4), brown (hair 5E4) to yellowish brown (bronze 5E5), towards the base brown (chestnut 6E7) to dark brown (chocolate 6F4), sometimes uniformly coloured in one of these colours. Lamellae 1–3 mm deep, L = 18–26 (30), l = 1–3, short decurrent, at first beige (4C3, ivory 4B3, putty 4B2), when mature dark grey brown (6F3 to 6F2), edges entire, concolourous or paler than the sides. Flesh 0.5–2 mm thick, elastic, with a strong naphthalene odour, light coloured (greyish-brownish).

Basidiospores (4.5)5.1–5.9(6.6) × (3.8)4.2–4.7(5.4) μm, av. 5.5 × 4.5 μm, Q (length/width) = (1.02)1.17–1.30(1.47), av. Q = 1.24, broadly ellipsoid, hyaline, smooth, inamyloid, not dextrinoid, thin-walled, hilar appendage 0.5–0.8(1) μm long. Basidia 4-spored, (32)38–48(58) × 6–7(–7.5) μm, av. 43.1 × 6.6 μm, hyaline, narrowly clavate, attenuated and flexuous toward base. Basidioles cylindrical to narrowly clavate, often flexuous, obtuse, ca. 22–40 × 3–5.5 μm. Pleurocystidia absent. Marginal cells on the lamellar edges not well differentiated, similar to basidioles on lamellar sides. Lamellar trama strongly intricate, subparallel to irregularly oriented, scarcely branched and irregularly inflated, hyphae 3–10(15) μm wide, composed of 20–100 μm long, fusiform or irregularly ventricose cells. Subhymenium pseudoparenchymatic, 15–25 μm deep, composed of branched, dense, 2–5 μm wide hyphae. Pileipellis near the pileus margin a hymeniderm, composed of cells arranged in one rank; terminal cells sphaeropedunculate, obpyriform or clavate, occasionally irregularly lobate, usually thin-walled, measuring (12)22–51(110) × (6)10–21.5(40) μm, av. 36.8 × 15.8 μm, Q = (0.98)1.32–3.71(8.5), av. Q = 2.5; subterminal cells usually cylindrical or fusiform and distinctly narrower but often also inflated, short and small cells (shorter than 10 μm) occasional, often bearing incrusted dark pigments, occasionally nodulose or with lateral projections, few branched, measuring (5)14.5–47(103) × (2.5)5–13.5(22) μm, av. 30.9 × 9.3 μm; terminal cells of hyphae in pileipellis near the pileus centre usually narrower, mainly clavate, often pedicellate and flexuous, measuring (11)20.5–46(83) × (5.5)7–16.5(28) μm, av. 33.4 × 12.8 μm, Q = (1)1.55–4.01(9.43), av. Q = 2.78; subterminal cells less frequently inflated and nodulose, often branched and flexuous, measuring (4)15–40(85) × (2)4–11(18) μm, av. 27.6 × 7.5 μm; pileus trama interwoven, 5–15 μm wide, subparallel, irregularly inflated and often branched, composed of up to 100 μm long cells, but often shorter than 20 μm. Caulocystidia fascicled, thin- or occasionally slightly thick-walled, repent or ascending, with terminal cells clavate, subcapitate or obpyriform, obtuse, often pedicellate and flexuous towards septum, measuring (12)26.5–61(149) × (4)6.5–11.5(19) μm, av. 42.8 × 8.9 μm; stipe trama of 5–12(15) μm wide, parallel hyphae, composed of 15–90(150) μm long cells. Clamp connections absent in all tissues.

Material examined: Czech Republic. Protected landscape area Blanský les, Chvalšiny, 9 September 2009, Z Egertová (SAV F-4443); Bílé Karpaty Mts., SE of the Vyškovec village, near the Ve Vlčí Nature Reserve (eastern edge), 24 October 2014, J Matouš (SLO673); Slovakia. Podunajská nížina lowland, SE of the Banka village, near the Koliba pod Ahojom, 24 September 2014, S Jančovičová (SLO770); ibid. 26 September 2014, S Jančovičová (SLO769); Sweden. Västra Götaland, Uddevalla, Ramseröd Nature Reserve, 7 October 2006, L and A Stridvall LAS06/123 (GB0060378); United Kingdom. England. Oxfordshire, Cothill Fen NNR, 26 October 2012, JA Webb (SAV F-4820); ibid. 27 October 2012, JA Webb (SAV F-4821); N Ireland. Tyrone, Omagh, Mountjoy Presbyterian Church, 5 November 2014, SE Evans [K(M)127075)]; Wales. Breconshire, Powys, Clydach, 5 November 2005, BM Spooner [K(M)135847]; Carmarthenshire, Brecon Beacons National Park, Brest CWM Llwyd, 22 October 2006, SE Evans [K(M)142036]; Breconshire, Mynydd Epynt, 2 October 2014, SE Evans (SAV F-4448); ibid. 24 October 2014, PJ Roberts (SAV F-4446); ibid. 5 November 2014, PJ Roberts (SAV F-4447).

Hodophilus tenuicystidiatus Jančovičová, Adamčík & Looney, sp. nov.

Figs. 8–9, 14, 18, 22, 26

MycoBank No.: MB818291

Etymology: the epithet refers to the narrow caulocystidia

Holotypus: SLO725

Diagnosis: Pileus fresh or when wet pale brown with a paler margin, becoming pale orange-grey to brown orange when dry, often rugose especially towards the centre; lamellae moderately close (L = 20–34); stipe apex with pale floccules, flesh with a strong naphthalene odour; caulocystidia crowded in large patches, mainly less than 7 μm wide (20.5–43 × 4–7 μm), flexuous, often with lateral nodules or projections; pileipellis a hymeniderm, terminal cells near the pileus margin sphaeropedunculate, subglobous, obpyriform or broadly clavate, subterminal cells of hyphae near the pileus margin rarely inflated and wider than 8 μm.

Pileus 8–16 mm broad, hemispherical, convex to plano-convex, usually depressed near the centre; margin inflexed to straight, crenate, when wet translucently striated halfway to the disc; surface matt, at first smooth but soon becoming rugose or veined especially towards the centre, hygrophanous; fresh or young basidiomata brown orange (blonde 5C4) or brown (hair 5E4) and towards the margin pale orange (5A3), grey-orange (5B3) or dull yellow (3B3), when dry orange-grey (5B2) to brown orange (5C3). Stipe 20–40 × 2–4 mm, usually flexuous, cylindrical and narrowed towards the base, sometimes compressed or longitudinally grooved, occasionally gnarled, smooth, shiny, apex with pale floccules, at the base white tomentose at times, near lamellae usually grey-orange (5B3) to brown (6D3), near the base light brown (blonde 5D4), yellowish brown (bronze 5E5), dark brown (chocolate 6F4) or dark grey-brown (6F3), sometimes uniformly coloured in one of these colours. Lamellae 1–3 mm deep, moderately close, L = 20–34, l = 1, sometimes anastomosed, short to deeply decurrent, brownish orange (5C3), grey brown (nougat 5D3), edges entire, concoloured. Flesh 0.5–1 mm thick in half radius of the pileus, elastic, with a strong naphthalene odour but also with unpleasant mercaptan components, orange grey (5B2).

Basidiospores (4.3)4.9–5.7(6.5) × (3.5)3.8–4.5(5) μm, av. 5.3 × 4.2 μm, Q = (1.04)1.19–1.36(1.54), av. Q = 1.28, broadly ellipsoid to ellipsoid, hyaline, smooth, inamyloid, not dextrinoid, thin-walled, hilar appendage up to 0.6-0.8 μm long. Basidia 4-spored, (29)33.5–39(42) × (5)5.5–6.5 μm, av. 36.3 × 6.2 μm, hyaline, narrowly clavate, attenuated and flexuous toward the base. Basidioles cylindrical to narrowly clavate, obtuse, often flexuous, ca. 2.5–5.5 μm wide. Pleurocystidia absent. Marginal cells on the lamellar edges not well differentiated, similar to basidiola (sterile immature basidia in hymenium) on lamellar sides. Lamellar trama composed of sub-parallel hyphae of mainly ventricose-fusiform cells, 4–15(20) μm wide, septa usually less distant than 100 μm (often <50 μm). Subhymenium pseudoparenchymatic, 15–25 μm deep, composed of branched, dense, 2–5 μm wide hyphae, gradually passing to underlying hyphae of the trama. Pileipellis near the pileus margin a hymeniderm, composed of cells arranged in one or two ranks; terminal cells sphaeropedunculate, subglobous, obpyriform or broadly clavate, measuring (9.5)18–38(63) × (4.5)6.5–17.5(34) μm, av. 28.1 × 12 μm, Q = (1.06)1.51–3.72(7.83), av. Q = 2.61; subterminal cells mainly narrow and cylindrical, occasionally also laterally inflated or nodulous, occasionally branched, measuring (2.5)10–33(65) × (2)3.5–10(20) μm, av. 21.5 × 6.6 μm. Hyphal terminations in pileipellis near the pileus centre occasionally with yellowish brown intracellular pigments, occasionally also with thick-walled elements, with terminal cells sphaeropedunculate or clavate, less frequently obpyriform, often lobate or undulate, occasionally with lateral projections, measuring (6)16–41.5(76) × (5)6–15.5(33) μm, av. 28.8 × 10.8 μm, Q = (0.71)1.51–4.3(9.67), av. Q = 2.9; subterminal cells more frequently flexuous, nodulous or with lateral projections, some with darker incrusted pigments, usually not branched, measuring (2)9.5–34(80) × (2)3–8.5(24) μm, av. 21.9 × 5.8 μm. Pileus trama of interwoven, 5–20 μm wide, subparallel, irregularly inflated and often branched hyphae, also with few narrow (3–5 μm) and pigmented hyphae, composed of up to 100 μm long cells, but often some shorter than 50 μm. Caulocystidia crowded in large patches, intricate, very variable in shape, mainly narrow subcylindrical or narrowly clavate, some wider, broadly clavate, usually thin-walled, often very flexuous, occasionally nodulous or with projections, usually with darker or sometimes very dark intracellular or parietal pigments, measuring (14)20.5–43(74) × (2)4–7(13) μm, av. 31.8 × 5.5 μm. Stipe trama of 3–12(18) μm wide, parallel hyphae, composed of 20–100 μm long cells (few >100 μm). Clamp connections absent in all tissues.

Material examined: Denmark. NW Jutland, Skive, Marienlyst Strand, 2 September 2007, D. Boertmann DEN-17 (CFMR7071); Estonia. Saaremaa, Viieristi protected area, 20 September 2008, S Adamčík (SAV F-2180); France. Oise: Sacy-le-Grand, marais de Sacy, 29 October 2014, P Clowez PAM14102901 (LIP); Slovakia. Oravské Beskydy Mts., Oravská Polhora village, Slaná voda, 9 October 2012, N Rybáriková (SAV F-3850); Podunajská nížina lowland, SE of the Banka village, near the Koliba pod Ahojom, 26 September 2014, S Jančovičová (SLO772, SLO780); Malé Karpaty Mts., NNW of the Plavecké Podhradie village, 9 October 2014, S Jančovičová (SLO725); United Kingdom. England. Oxfordshire, Hinksey Heights Fen, 26 September 2010, JA Webb (SAV F-4822); Northamptonshire, King’s Cliffe, Ring Haw, 23 October 2013, J Blincow [K(M)189409]; Wales. Pembrokeshire, Somerton farm, 14 November 2010, D Harries (SAV F-4440).

Hodophilus subfoetens Adamčík, Jančovičová & Looney, sp. nov. Figs. 10, 15, 19, 23, 27

MycoBank No.: MB 818292

Etymology: the species is named based on pileus colour resembling Ho. foetens

Holotypus: SAV F-3488

Diagnosis: Pileus with brown colours that become darker brownish black when mature or dry, lamellae relatively distant (L = 15–20); stipe only slightly pruinose near lamellae, flesh with strong naphthalene odour; caulocystidia obtuse or apically constricted, usually not flexuous, clavate or subcylindrical, mainly shorter than 30 μm and moderately wide (15.5–36.5 × 5–9.5 μm); pileipellis a hymeniderm, terminal cells near the pileus margin clavate or obpyriform, subterminal cells of hyphae near the pileus margin rarely inflated and wider than 8 μm, terminal cells of hyphae near the pileus centre relatively narrow (length/width ration >2.5).

Pileus 6–18 mm broad, convex, weakly depressed near the centre when old, margin striated at 2/3 when wet, sometimes slightly crenulated, surface smooth, near the centre rugulose, matt, hygrophanous, colour when wet brownish grey to grey brown (5E3, hair brown 5E4, drab 6D2), when dry brownish black (nutria brown 6D2). Stipe 20–30 × 1–1.5 mm, cylindrical and usually narrowed near the base, smooth and shiny except where slightly pruinose near lamellae, concolourous with the pileus. Lamellae 1–2 mm wide, L = 15–20, l = 0–1, short decurrent, grey brown (bronze 5E5) or chocolate brown (6F4). Flesh elastic, with a naphthalene odour.

Basidiospores (4.6)5–5.7(6.4) × (3.3)3.9–4.5(5) μm, av. 5.3 × 4.2 μm, Q = (1.08)1.20–1.36(1.53), av. Q = 1.28, broadly ellipsoid to ellipsoid, hyaline, smooth, inamyloid, not dextrinoid, thin-walled, hilar appendage 0.5–0.9 μm long. Basidia 4-spored, (28)32.5–39(41) × (5)5.5–6.5 μm, av. 35.6 × 5.9 μm, hyaline, narrowly clavate, attenuated and slightly flexuous toward base. Basidioles cylindrical to narrowly clavate, often flexuous, obtuse, 3–5.5 μm wide. Pleurocystidia absent. Marginal cells on the lamellar edges not well differentiated. Lamellar trama of intricate, subparallel or irregularly oriented, often narrowed near septa, composed of fusiform or irregularly ventricose cells, mixed with a few narrow and cylindrical, 5–10 (25) μm wide and up to 100 μm long. Subhymenium pseudoparenchymatic, 15–25 μm deep, composed of branched, dense, 2–5 μm wide hyphae. Pileipellis near the pileus margin a hymeniderm, composed of cells arranged in one rank; terminal cells clavate or obpyriform, thin-walled, measuring (16)22.5–43(64) × (6)9–17.5(38) μm, av. 32.9 × 13.3 μm, Q = (0.9)1.45–4.09(12), av. Q = 2.77; subterminal cells usually narrow, cylindrical, not branched, not flexuous, measuring (3.5)13.5–35(47) × (2)3.5–10(22) μm, av. 24.3 × 6.7 μm. Pileipellis elements near the pileus centre similar to those near the margin, terminal cells of hyphae often narrower and/or longer, measuring (15)23–47.5(73) × (5.5)7.5–16(25) μm, av. 35.4 × 11.8 μm, Q = (1.07)1.91–4.5(10), av. Q = 3.21; subterminal cells similar to those near the pileus margin, measuring (4)11.5–37.5(66) × (2)3–8.5(22) μm, av. 24.6 × 5.7 μm. Pileus trama of interwoven, 3–20 μm wide, subparallel to irregularly oriented hyphae, composed of fusiform, ventricose or some dispersed sub-cylindrical, up to 100 μm long (frequently even shorter than 20 μm) cells. Caulocystidia dispersed or in relatively small fascicles, repent or with ascending tips, thin-walled, clavate or subcylindrical, usually not flexuous, obtuse or apically constricted, measuring (11)15.5–36.5(75) × (3)5–9.5(14) μm, av. 26 × 7.2 μm. Stipe trama of 3–10 μm wide, parallel, cylindrical, entire hyphae, with septa usually not more than 100 μm distant. Clamp connections absent in all tissues.

Material examined: Slovakia. Záhorská nížina lowland, Abrod National Natural Reserve, 30 September 2002, S Adamčík (SAV F-3488); Biele Karpaty Mts., Krivoklátske lúky Nature Monument, 31 July 2005, S Adamčík (SAV F-3497); Biele Karpaty Mts., Blažejová Nature Monument, 27 September 2005, S Jančovičová, (SAV F-3490); Laborecká vrchovina Mts., Jaruchy, 12 October 2005, J Terray (SAV F-3487); United Kingdom. Wales. Monmouthshire, Abergavenny, The Blorenge, 5 November 2005, R Shotbolt [K(M)135850].

Hodophilus pallidus Adamčík, Jančovičová & Looney, sp. nov. Figs. 11–12, 16, 20, 24, 28

MycoBank No.: MB 818294

Etymology: the epithet refers to pale colour of basidiomata

Holotypus: SAV F-3486

Brief diagnosis: Pileus orange-grey to greyish orange, when dry pale brownish orange; lamellae distant (L = 10–17); stipe near lamellae with pale, fine granulations, flesh with strong naphthalene odour; caulocystidia obtuse, fascicled or isolated, clavate or subcylindrical, occasionally also with short ellipsoid terminal cells, mainly shorter than 30 μm and wider than 7 μm (14.5–39.5 × 5.5–11 μm); pileipellis a hymeniderm, terminal cells near the pileus margin subglobous or obpyriform, subterminal cells of hyphae near the pileus margin rarely inflated and wider than 8 μm, terminal cells of hyphae near the pileus centre usually broad and often subglobous (length/width ration mainly <2.5).

Pileus 6–18 mm broad, convex, margin slightly translucently striate when wet, slightly crenulated, inflexed and only straight when old, surface smooth, but wrinkled near the margin when old, matt, hygrophanous, when wet orange-grey (6B2 to 6B3) to greyish orange (5B2 to 5B3), when dry pale brownish orange (5A2). Stipe 25–40 × 1–2 mm, cylindrical and usually flexuous, smooth and near the lamellae with fine pale granulations, near the lamellae greyish orange (5B3) to orange grey (6B3), towards the base sometimes darker brownish orange (5C3), orange-brown to grey-brown (7C3-7E3). Lamellae 1–2 mm wide, distant, L = 10–17, l = (0)1, short decurrent, concolourous with the pileus surface, edges entire. Flesh elastic, with strong naphthalene odour.

Basidiospores (4.7)5.1–5.7(6.1) × (3.5)3.9–4.6(5) μm, av. 5.4 × 4.3 μm, Q = (1.02)1.17–1.36(1.49), av. Q = 1.27, broadly ellipsoid to ellipsoid, hyaline, smooth, inamyloid, not dextrinoid, thin-walled, hilar appendage 0.6–1.1 μm long. Basidia 4-spored, (27)30.5–38(45) × 5–6(6.5) μm, av. 34.3 × 5.6 μm, hyaline, narrowly clavate, attenuated and slightly flexuous toward base. Basidioles cylindrical to narrowly clavate, often flexuous, obtuse, 3–5.5 μm wide. Pleurocystidia absent. Marginal cells on the lamellar edges not well differentiated, but in average shorter than basidioles on lamellar sides. Lamellar trama of intricate, subparallel or irregularly oriented, near septa often narrowed, composed of fusiform or irregularly ventricose cells, mixed with a few narrow and cylindrical, 5–10(25) μm wide and up to 100 μm long. Subhymenium pseudoparenchymatic, 15–25 μm deep, composed of branched, dense, 2–5 μm wide hyphae. Pileipellis near the pileus margin a hymeniderm, composed of cells arranged in one rank; terminal cells subglobous or obpyriform, occasionally thick-walled, measuring (15)19.5–39(62) × (8)10.5–20(29.5) μm, av. 29.4 × 15.3 μm, Q = (0.95)1.21–2.95(5.3), av. Q = 2.09, usually regularly rounded and not pedicellate; subterminal cells usually narrow, cylindrical or narrowly fusiform, occasionally branched, rarely inflated, not flexuous or nodulous, measuring (2)12–35(55) × (2.5)3–10.5(18) μm, av. 24.3 × 6.7 μm. Pileipellis elements near the pileus centre similar to those near the margin, terminal cells of hyphae more frequently pedicellate but otherwise similar to those near the pileus margin, measuring (17)22.5–37(49) × (8.5)10.5–18(24) μm, av. 29.8 × 14.2 μm, Q = (1.02)1.5–2.93(4), av. Q = 2.22; subterminal cells similar to those near the pileus margin but more frequently inflated, measuring (5)13–32.5(47) × (2)3.5–8.5(12.5) μm, av. 22.8 × 5.9 μm. Pileus trama of interwoven, 3–20 μm wide, subparallel to irregularly oriented hyphae, composed of fusiform, ventricose or some dispersed sub-cylindrical, up to 100 μm long (frequently even shorter than 20 μm) cells. Caulocystidia fascicled, but some dispersed and isolated, mixed with dispersed basidia, repent or with ascending tips, thin-walled, clavate or subcylindrical, but occasionally also with short ellipsoid terminal cells, usually not flexuous, obtuse, terminal cells measuring (6)14.5–39.5(60) × (3.5)5.5–11(18) μm, av. 27 × 8.3 μm. Stipe trama of 3–10 μm wide, parallel, cylindrical, entire hyphae, with septa usually not more than 100 μm distant. Clamp connections absent in all tissues.

Material examined: Slovakia. Biele Karpaty Mts., Krivoklátske lúky Nature Reserve, 26 September 2005, S Adamčík (SAV F-3486); Biele karpaty Mts., Grúň Nature Monument, 26 September 2005, S Adamčík (SAV F-3489).

Discussion

Placement, delimitation and nomenclature of Ho. albofloccipes

The phylogenetic placement and naphthalene odour of Ho. albofloccipes constitute a similar pattern as in the North American species Ho. subfuscescens (A.H. Sm. & Hesler) Adamčík, Birkebak & Looney (Adamčík et al. 2016). They are both placed in the H. micaceus superclade apart from all other species with a naphthalene odour, which are grouped in the Ho. foetens superclade (Fig. 1). Species clades that include the types of those two species also contain both collections with and without a naphthalene or unpleasant odour. Both of these species bear olive and yellow-brown tints and both have frequent small subterminal cells in the pileipellis. One collection originally identified as C. foetens included in this study [K(M)161018] placed in another species clade (Ho. micaceus group 1) was probably identified based on the naphthalene odour. This suggests that sometimes this odour may also appear in various members of Ho. micaceus superclade, although probably not with a strong intensity and not persisting in the dry specimens. Hodophilus albofloccipes, despite its odour, is morphologically and genetically distinguished from other species in the Ho. foetens superclade. More challenging is the nomenclature of this species, because the species clade with the type of Ho. albofloccipes contains specimens without distinctive odour and any older name used for European Hodophilus species might be a synonym and the correct one. We think that the delimitation of Ho. albofloccipes requires observation and type studies of other taxa of the Ho. micaceus superclade, and for this reason we did not provide a description and other notes on our collection of this species.

Morphological delimitation and variability of species within Ho. foetens superclade

Our previous study (Adamčík et al. 2016) showed the significance of micromorphological characters for species delimitation, contrary to similar habit (field appearance) of North American taxa, that do not allow their identification in the field. This has been demonstrated in particular for Ho. heslerii Adamčík, Birkebak & Looney and Ho. smithii Adamčík, Birkebak & Looney. All North American taxa included in that study have been defined by a unique combination of micromorphological characters that allow the preparation of a key without using macromorphological characters. The morphological analysis of European Hodophilus members, however, does not show decisive differences among all species under the microscope. The most conspicuous and useful microscopic element seems to be morphology of caulocystidia. Hodophilus tenuicystidiatus has a maximum average value of caulocystidia width of 6.7 μm that only partly overlaps with Ho. subfoetens (minimum average width 6.6 μm) and does not overlap with the other two species at all (Table 2). In addition, the caulocystidia of Ho. tenuicystidiatus are flexuous and occasionally also nodulous or with lateral projections, while in Ho. subfoetens they are more regularly shaped. The shape and length of caulocystidia are also useful for differentiating Ho. foetens from Ho. subfoetens and Ho. pallidus. All three species have a similar width of caulocystidia (but they are usually narrower and on average up to 7.5 μm in Ho. subfoetens). Hodophilus subfoetens and Ho. pallidus differ from Ho. foetens especially by usually not having flexuous caulocystidia that often have short terminal cells with average lengths up to 31 μm, in contrast to a minimum of 31.6 μm for Ho. foetens. Hodophilus subfoetens and Ho. pallidus, however, do not show any conspicuous differences under the microscope. The caulocystidia of Ho. subfoetens are often apically constricted (Fig. 15), while in Ho. pallidus they are always obtuse-rounded (Fig. 16). This character, however, was not conspicuous and consistent in all studied collections. The distinct difference we observed in the shape of terminal cells of hyphae near the pileus centre that are narrower and with length/width ratio always above 2.7 in the first but up to 2.5 in the second species. However, we think that the reliability of this character should be checked on a larger sampling (only two collections of Ho. pallidus studied).

Field characters delimit pairs of the studied European species. Hodophilus foetens and Ho. tenuicystidiatus have usually more than 20 lamellae and the other two species, Ho. subfoetens and Ho. pallidus, have a maximum 20 lamellae (not including short lamellulae). Lamellae number or spacing has not been used for species delimitation within the genus Hodophilus, but is used in various groups of small agarics (e.g. Adamčík and Jančovičová 2011). A useful character is also pileus colour that becomes dark brown to black brown with age or drying in Ho. foetens and Ho. subfoetens, while it remains pale brown or becomes even paler brownish in the other two species. Dark brownish-black spots or areas on the surface of basidiomata of Ho. foetens are particularly conspicuous and typical. The number of lamellae was also observed on each individually studied basidiomata in dried material (under a stereo microscope) and, in our opinion, our data are statistically sufficient to recognise the species pairs. The pileus colour of Ho. subfoetens and Ho. pallidus, however, is described based on a limited number of observations and we recommend combining this character with micromorphological features discussed above.

Ecology and distribution

This study confirms the conclusion of Adamčík et al. (2016) that, contrary to North American Hodophilus taxa reported mainly from forest habitats, European samples reported prior to or during this study were collected mainly in grasslands or bushy forest margins. The core of our European collections of Hodophilus with a naphthalene odour have been sampled from Slovakia and the United Kingdom. Both these countries have representatives of all four species clades except Ho. pallidus, which was collected only in Slovakia. Moreover, Ho. foetens and Ho. tenuicystidiatus samplings also contain Scandinavian collections, and these two species are probably well distributed in temperate and boreal areas of Europe. It is not possible to conclude much about the distribution of Ho. subfoetens and Ho. pallidus based on available data. Slovakian data show that all four species may co-occur in one relatively small area. Even at a single site, more than one species may be present: Ho. foetens (SLO769, SLO770) and Ho. tenuicystidiatus (SLO772, SLO780) were collected during one day (26 September 2016) at a single site near the Banka village, and Ho. subfoetens (SAV F-3497) and Ho. pallidus (SAV F-3486) were both collected at Krivoklátske lúky Nature Manument (Supplementary Table 1).

Hodophilus with a naphthalene odour in European literature

The morphological descriptions of Ho. foetens in the current European literature used to be limited to spore dimensions and some brief information about pileipellis or other micromorphological characters. Such data on the microscopy of Ho. foetens are not reliable to compare with species differences described here, and we must rely only on macromorphological characters when trying to understand a concept of the species.

A major part of European authors followed the species concept of Ho. foetens presented in the original diagnosis (Phillips 1878) and described the pileus colour as dark brown (Lange 1940; Moser 1967; Printz and Silvertsen 1992; Horak 2005). Other authors are less specific in the interpretation of the brown colour of the pileus and their descriptions may encompass all four species described here (Bresadola 1928; Heim 1969; Bon 1977). The darker blackish colour that develops during maturation of basidiomata are mentioned only by Boertmann (2012) in the key to Nordic species of the genus Camarophyllopsis. Although almost all European mycologists (except for Kovalenko et al. 2012) follow the wide concept of Ho. foetens as a single species with a naphthalene odour, their descriptions are sometimes probably based on material of one species in a narrow-concept only. Ghyselinck (2003) published a photo of Ho. foetens that shows dark spots on the pileus and moderately close gills typical for Ho. foetens. Arnolds (1990) described this species as having a brown pileus becoming paler when dry and 12–22 lamellae, which corresponds to our description of Ho. pallidus. Kovalenko et al. (2012) described the pileus of Ho. foetens as having tobacco brown, yellow-brown and grey-brown colours becoming paler when dry and relatively wide caulocystidia, which may correspond to Ho. pallidus. In conclusion, European data about the distribution of Ho. foetens needs urgent revision and most probably contains information about more than one species.

Notes on Hodophilus species without a naphthalene odour

Based on our knowledge and unpublished type studies, five species of the genus Hodophilus without a strong naphthalene odour are widely accepted in the European literature (treated in the genus Camarophyllopsis). European keys to identification of Camarophyllopsis accept three (e.g. Arnolds 1990; Boertmann 2012) to five (Bon 1977) Hodophilus species without this odour. Hodophilus atropunctus is the only species accepted in every general European publication of the genus, a species identified by its darker scales or granulations on the stipe. Collections with such a stipe surface are placed in our phylogeny in two different clades within both Hodophilus superclades (one clade labelled as Ho. micaceus group 2 in Fig. 1 because it also contains collections without darker scales or granulations), but we do not have enough information to judge which one corresponds to the typical Ho. atropunctus. The remaining species without a naphthalene odour are recognised based on the colour of the stipe and lamellae. Some authors (Bon 1977; Horak 2005; Boertmann 2012) also reported North American species Ho. hymenocephalus from Europe, but our tree (Fig. 1) shows no European collection in the Ho. hymenocephalus clade and suggests this species does not occur in Europe. Three remaining species names, Ho. micaceus, C. phaeoxantha (Romagn.) Arnolds and C. phaeophylla (Romagn.) Arnolds (the latter two not combined yet in the genus Hodophilus), might correspond to any of the remaining European clades of Hodophilus without a naphthalene odour (labelled as Ho. micaceus group 1-3 and C. phaeoxantha group 2 in Fig. 1).