Mycol Progress DOI 10.1007/s11557-013-0938-y

ORIGINAL ARTICLE

Epitypification of Hebeloma crustuliniforme Jan Vesterholt & Ursula Eberhardt & Henry J. Beker

Received: 17 June 2013 / Revised: 18 September 2013 / Accepted: 25 September 2013 # German Mycological Society and Springer-Verlag Berlin Heidelberg 2013

Abstract It is widely recognised that there is no consistent use of the name H. crustuliniforme. The name has been used to represent a number of different taxa and indeed taxa from sections of Hebeloma that are morphologically and molecularly well separated. The holotype of H . crustuliniforme is plate 308 Bulliard from 1787 and while it can be interpreted, no such interpretation can be without ambiguity. It is clear from existing literature that modern authors have applied numerous different interpretations to this name and no real consensus exists. Indeed it appears that at various times most of the medium to large species within both sections Denudata and Velutipes Vesterh. have been referred to as H . crustuliniforme. Within this paper an epitype is selected for H. crustuliniforme in order to give the taxon a precise meaning and a detailed species description is given. Molecular data combined with the results of intercompatibility tests of Aanen and Kuyper published earlier support the definition of H . crustuliniforme adopted in this paper as a distinct taxon and as a biological species. We strongly recommend that this taxon be referred to as Hebeloma crustuliniforme (Bull.) Quél. emend. Vesterh., U. Eberh. & Beker in order to emphasise that it is the specific taxon rather than the complex to which it is being referred.

Jan Vesterholt Deceased. Electronic supplementary material The online version of this article (doi:10.1007/s11557-013-0938-y) contains supplementary material, which is available to authorized users. J. Vesterholt Natural History Museum of Denmark, Gothersgade 130, DK-1123 Copenhagen, Denmark U. Eberhardt (*) Staatliches Museum für Naturkunde Stuttgart, Rosenstein 1, D-70191 Stuttgart, Germany e-mail: [email protected] H. J. Beker Rue Père de Deken 19, B-1040 Bruxelles, Belgium

Keywords Taxonomy . Systematics . Ectomycorrhizal fungi . Agaricales . Non-cortinate . MCM7

Introduction Hebeloma spp. are widespread ectomycorrhizal fungi in the northern hemisphere, and specifically across Europe, occurring in a wide range of different habitats (Marmeisse et al. 1999). As a result of their morphological similarity and plasticity, their determination to species level is difficult. Hebeloma crustuliniforme, the type species of the long accepted sect. Denudata (Fr.) Sacc, is probably the most often recorded Hebeloma sp. For example the Fungal Records Database of Britain and Ireland (http://www.fieldmycology. net/FRDBI/FRDBI.asp) has only one Hebeloma sp., namely H. crustuliniforme, in its list of fungal species most recorded. (At the time of writing (October 2012) there are 3080 records for H. crustuliniforme out of a total of 8227 Hebeloma records, i.e. 37 %.) Unfortunately there appears to be little consensus for what is represented by the name H. crustuliniforme. For example we have examined Bruchet’s collections of H . crustuliniforme and found them to be different species from different sections (sect. Velutipes and sect. Denudata). Although we have not been able to examine authentic material, the images of Bon (2004), Courtecuise and Duhem (1995) and Cetto (1976) appear largely to represent H. leucosarx (P.D. Orton) emend. Grilli (synonymous with H. velutipes Bruchet) (Grilli 2007). The photograph in Breitenbach and Kränzlin (2000) of H. crustuliniforme is likely to represent either H. leucosarx or H. stenocystis J. Favre ex Quadr., both from sect. Velutipes. On the other hand authors such as Dähncke (1993), Vesterholt (2005), Gerhardt (2003), Ryman and Holmåsen (1992), Knudsen (2003), and Boccardo (2008) show H. crustuliniforme as a Hebeloma with a pale cap and a very floccose stem that represents a complex of species from sect. Denudata. We have examined material of Moser published in (Moser and Jülich 1998) to which he referred as H. crustuliniforme and found this

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to be a taxon close to H. eburneum but with much more slender basidiomes and with smaller spores. Our own experience of receiving a number of collections labelled H. crustuliniforme is that this has represented 13 different taxa. We have also examined all the GenBank and UNITE records labelled H. crustuliniforme. There are 23 ITS sequences labelled Hebeloma crustuliniforme or from material labelled such deposited in public databases (GenBank, UNITE (http://unite. ut.ee; Abarenkov et al. 2010), CBS Strain Database (http:// www.cbs.knaw.nl/collections/Biolomics.aspx?Table=CBS% 20strain%20database) of 12 Dec 2012) which, based on sequence data, we believe to represent at least 10 different taxa. Similarly examining photographs of H. crustuliniforme available on the World Wide Web we find a number of different Hebeloma species are represented. Our own personal experience of examining herbarium material labelled H . crustuliniforme is that it is likely to represent almost any medium to large species from either sect. Denudata or Velutipes. Bulliard’s (1787, pl. 308) original plate (which is the holotype under Art. 9.1 of the nomenclatural code (McNeill et al. 2012) of Agaricus crustuliniformis shows a fairly slender species with distinctly brown cap. It cannot be said with certainty which species this icon represents. It may be conspecific with either species from sect. Denudata or sect. Velutipes. It is likely to be a species that would have occurred around Paris where Bulliard lived and worked. In the protologue, Bulliard (1787) described it as “trés commune en automne dans le bois, les prairies”. However, this would also apply to a number of species. Most likely, his concept of A. crustuliniformis included more than one taxon, and a later plate (Bulliard 1792, pl. 546) most likely represents a different brown-capped Hebeloma species, perhaps H. quercetorum Quadr. from sect. Velutipes. However Bulliard’s original plate (308) is unlikely to represent a species from sect. Velutipes as these taxa do not normally have a floccose stipe; the stipe is usually velutinous or pruinose near the apex at most. It is more likely to represent one of the medium to large species from sect. Denudata which would then account for the floccose stipe shown on the plate. While these species are often paler coloured, darker coloured collections do exist. The important point here is that no determination based on Bulliard’s original plate is unambiguous and all such determinations are debateable. Aanen and Kuyper (1999, 2004) combined all the medium to large taxa within sect. Denudata under the name H . crustuliniforme. They opted for a species concept that—in the absence of clear morphological differences separating candidate taxa—allowed species to be paraphyletic in molecular analyses and did not require all members of a species to be biologically compatible in mating tests (Aanen and Kuyper 1999). As a consequence, their concept of H . crustuliniforme included members of five intercompatibility groups (ICG) some of which could be distinguished from each other morphologically. This

concept of H. crustuliniforme will be referred to as Hebeloma crustuliniforme complex. In order to resolve the situation of H. crustuliniforme and provide nomenclatural stability, we believe the soundest option is to select an epitype for Hebeloma crustuliniforme that ties it to a single taxon. Art. 9.8 (McNeill et al. 2012) says: 9.8. An epitype is a specimen or illustration selected to serve as an interpretative type when the holotype, lectotype, or previously designated neotype, or all original material associated with a validly published name, is demonstrably ambiguous and cannot be critically identified for purposes of the precise application of the name of a taxon. The taxon to which it is epitypified should preferably be one of the taxa that has commonly been referred to in the past as H . crustuliniforme and which is also sufficiently matched to Bulliard’s original plate (1787, pl. 308) that it is the most likely taxon that Bulliard intended. A disadvantage of this approach is that there may be confusion with regard to collections bearing the name Hebeloma crustuliniforme and such problems may persist into the future as any records or material that did not bear an “emend.” designation would leave uncertainty as to whether the determination was based on the wide view of the taxon or a narrow view. An alternative approach may be to try and reject the name H . crustuliniforme totally (under Article 56.1, McNeill et al. 2012), but there would be a danger of such a proposal being rejected and the use of the name continuing. With no epitype selected, the current situation would continue where the name is applied to various taxa. Further, since H. crustuliniforme is the type species of sect. Denudata, the disappearance of the name would require the selection of a new type for this section. We believe that Art. 9.8 was designed and intended for exactly this situation, whereas, albeit appealing, to propose a rejection of the name under Art. 56.1 would be an undesirable course, because the name would probably remain in circulation for a long time. As a consequence of these arguments, we have chosen what we consider to be the correct interpretation of the Code and the more pragmatic way forward and selected an epitype to link this name with a specific taxon. We strongly recommend that this taxon be referred to as Hebeloma crustuliniforme (Bull.) Quél. emend. Vesterholt, U. Eberhardt & Beker in order to emphasise that it is the specific taxon rather than the complex to which it is being referred. The epitypification of H. crustuliniforme is a first step towards the goal of teasing the H. crustuliniforme complex apart and giving new descriptions of the species in sect. Denudata (Eberhardt et al. in prep.). In order to demonstrate that the taxon H . crustuliniforme in the sense of the epitypification is indeed a distinct species, we performed molecular analyses based on three loci, the ITS and

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two single copy genes, and have included representatives of diverse representatives of the H. crustuliniforme complex, i.e., H . crustuiniforme sensu Aanen and Kuyper (2004) complex and closely related sequences.

Materials and methods A set of 49 collections was selected, representing the Hebeloma crustuliniforme complex and related taxa. As outgroup H . helodes p.p. sensu (Aanen and Kuyper 2004) was chosen. The outgroup relationship was shown by Aanen and Kuyper (2004) and further supported by our own studies with additional genes (not published). The molecular analysis is based on three loci, ITS, RPB2 and MCM7 (a DNA replication licensing factor). The ITS data set is complete. For some collections, particularly older collections, single copy genes could not be amplified. Details and GenBank accession numbers are given in Table 1. Working with Hebeloma spp., ITS-PCR of some DNA extracts result in amplicons with length variation and single nucleotide polymorphisms apparent in direct sequencing, suggestive of two different types of ITS. These results are highly reproducible, even when starting from different DNA extracts of the same collection. Rare deviations from this rule have only been observed when different fruit bodies of the same collection were used for DNA extraction. The proportion of any sequence type in a DNA extract must be relatively high in order to show in direct Sanger sequencing results from PCR products. Therefore we assume that these observations are evidence of intragenomic variation, possibly due to the presence of deviating maternal and paternal alleles (Aanen et al. 2001), and not due to technical problems. In such cases, both copies were submitted to GenBank and used in single-locus ITS analyses. Molecular methods used have been described in detail in a recent publication (Eberhardt et al. 2013). New MCM7 primers for basidiomycetes were constructed based on details published by (Schmitt et al. 2009), namely bMcm7-709for (ACI CGG IGT RTC IGA RGT MAA RCC) and bMcm71348rev (GAY TTS GCX ACM CCI GGR TCR CCC AT). DNA alignments were done in Mafft (version 7, as implemented on http://mafft.cbrc.jp/alignment/server/, Katoh and Standley 2013) with the ‘FFT-NS-I’ strategy for ITS and the ‘Auto’ strategy for RPB2 and MCM7 (Katoh et al. 2002). The alignments were checked for alignment consistency. Data matrices of different loci were merged in Mesquite (version 2.75, Maddison and Maddison 2011, http://mesquite project.org). Compatibility of data sets stemming from different loci was tested following the principle of Kauff and Lutzoni (2002), with a threshold of 70 %. ML analyses were performed in RAxML (as implemented on http://phylo.org (Fig. 1) or http:// phylobench.vital-it.ch (electronic supplementary material), Stamatakis 2006; Stamatakis et al. 2008; Miller et al. 2010) for

Table 1 Sequence accession numbers. Herbaria BR – Meise, Belgium, C – Copenhagen, Denmark, GLM – Herbarium Senckenbergianum Görlitz, Germany, K – Kew, U.K., PDD – New Zealand Fungal and Plant Disease Herbarium, Auckland, New Zealand, WBS – Wijster Research Station Herbarium, Netherlands, now part of L – Leiden, Netherlands. Private collections: HJB – Henry J. Beker, GC – Gilles Corriol. * – Hebeloma crustuliniforme (Bull.) Quél. emend. Vesterh., U. Eberh. & Beker (= Hebeloma crustuliniforme s.str.). $ – epitype. º – Outgroup (H. helodes sensu Aanen and Kuyper 2004) HJB collection Other collection: ITS and database collection number GenBank reference acc. no.

RPB2 GenBank acc. no.

MCM7 GenBank acc. no.

HJB11051

JN943865

KF309463 KF309496

HJB11100

JN943867

KF309442 KF309482

JN943869

KF309444 KF309484

HJB11133 *HJB10832

HJB:JV91-670

*HJB10902

HJB:JV00-643

KF309407

*HJB10977

GLM:GL08704

KF309408

*HJB11215

KF309409, KF309445 KF309485 KF309410 JN943870 KF309440 KF309480

*HJB11237 *HJB12244

KF309406

GLM:HBo79139

*HJB12264

KF309419

KF309451

KF309418

KF309452

*HJB12292

HJB:JV91-669

KF309396

*HJB12481

L:WBS9689

KF309413

*HJB12803

L:WBS9691

KF309414

KF309491

*HJB12807

L:WBS9581

KF309415

KF309454 KF309492

*HJB12811

L:WBS9546

KF309453

KF309416

KF309455 KF309493

*HJB12815

JN943849

KF309459

*HJB12842

JN943848

KF309460

KF309423

KF309461

*HJB12851

HJB:MAK 08-10015

*HJB13430

KF309417

*HJB9053

KF309398, KF309427 KF309399 JN943881 KF309429 KF309469

*HJB9876 *HJB9930

K:K(M)108529

KF309397

*HJB13713

§

KF309424

BR:BR-MYCO 173990–69 epitype, C: C-F-90146”

HJB10525 HJB11687 HJB12769

HJB:JV08-451

KF309462 KF309495

JN943876, KF309436 KF309476 KF309401 JN943863, KF309447 KF309487 KF309412 JN943850 KF309458

HJB9267

JN943880

KF309428 KF309468

ºHJB11328

JN943864

KF309446 KF309486

ºHJB8098

JN943879

KF309426 KF309467

ºHJB9819

JN943871

KF309439 KF309479

HJB11079

JN943866

KF309441 KF309481

HJB11107

JN943868

KF309443 KF309483

HJB12007

JN943857

KF309449 KF309488

ºHJB11728

JN943862

KF309464 KF309497

ºHJB11716 ºHJB12107 ºHJB12460

GC:GC 07 09 29 03 L:WBS9508

JN943861

KF309448

JN943854

KF309450 KF309489

JN943858

Mycol Progress Table 1 (continued) HJB collection Other collection: ITS and database collection number GenBank reference acc. no.

RPB2 GenBank acc. no.

MCM7 GenBank acc. no.

ºHJB12730

KF309422

KF309457

HJB10384

KF309400

KF309432 KF309472

KF309404

KF309498 KF309465

KF309405

KF309438 KF309478

HJB10776 HJB10833

HJB:JV96-341

HJB12655

JN943854, KF309466 KF309490 KF309420 JN943877 KF309431 KF309471

HJB10282 HJB10692

PDD:PDD102994 JN943874

KF309437 KF309477

HJB10446

JN943875

KF309435 KF309475

HJB10260

KF309403

KF309430 KF309470

HJB10422

JN943878

KF309433 KF309473

HJB10427

KF309402

KF309434 KF309474

KF309421

KF309456 KF309494

HJB12323

C:JV08-278

all loci separately and for a merged matrix, with and without separate data partitions for different loci and codon positions. Trees were visualized using FigTree (version 1.4.0, Rambaut 2012, http://tree.bio.ed.ac.uk/software/figtree/.) Distance values of ITS data were calculated in PAUP* (Swofford 2002) with two different distance measures, (1) absolute distance of DNA data and discounting gap information and (2) total character difference, with the gap character forming a character state in addition to the IUPAC DNA characters. Distance measure (1) gives results comparable to Jukes Cantor distances, whereas in (2) all possible differences are considered. Morphological methods have been described in detail by Eberhardt et al. (2013). When measuring cheilocystidia at the gill edge it is of utmost importance not to be selective (Vesterholt 2005). Microscopic measurements are based on a large number of elements, i.e. 50 spores, 100 cheilocystidia apices and 20 cheilocystidia with view to the other measurements. In the description, both mean and median are noted to give an indication whether the measurements follow a normal distribution.

Results Figure 1a shows the ML result of an analysis of the ITS sequences generated for this study in combination with the ITS sequences published by D. Aanen (AF124665, AF124668, AF124671, AF124672, AF124678, AF124681, AF124683, AF124687, AF124688, AF124694, AF124696, AF124706, AF124708, AF124716), covering Hebeloma crustuliniforme sensu Aanen and Kuyper (2004), and its closest relatives in terms of ITS. This figure was included to demonstrate that the material used for this study is representative of the

H. crustuliniforme complex and the selected outgroup. As before in Aanen and Kuyper (2004) the H. crustuliniforme complex is paraphyletic. The sequences of nineteen collections (2004) including four collections of D. Aanen representing ICG5 form a clade with bootstrap support of 89 % (collection WBS9581 was sequenced twice, once by D. Aanen and by us). This clade contains the collection chosen as epitype for H. crustuliniforme and is consequently labelled in Fig. 1a as H. crustuliniforme s.str. Furthermore, the clade contains both ITS copies of length-deviant ITS-types encountered in two of the collections clustering in this clade. The length deviation consisted of one copy of the ITS showing unique insertions of one or four base pairs, respectively, unique not only in the context of the species, but also of the alignment. To further investigate the monophyly of this clade, an analysis of three genetic regions combined was done on the 49 collections analysed for this study. The alignments included 466 positions for the ITS (spacer regions only), 733 positions for RPB2 and 668 positions for MCM7. Results of single locus analyses are in the electronic supplementary material to this article. Different data treatment variants resulted in almost identical ML topologies (results not shown). Only a single conflict occurred among the ML topologies of single loci, concerning the sister clade relationship of clade A (clade identifiers see Fig. 1b and in Figs. 1 and 2 of the electronic supplementary material) with clade C in the RPB2 topology and with clade B in the MCM7 topology. This conflict was considered immaterial for the question at hand, and the alignments were thus concatenated. Figure 1b shows the best ML result of the three-loci-alignment, calculated with nine data partitions, one for the ITS and four for the coding genes each, for the three codon positions and one intron partition. Bootstrap analyses were done with 700 replicates. Further fine-tuning of the analysis was considered superfluous as the corresponding analysis with just a single data partition (result not shown) produced the same topology with very similar bootstrap support values. All sequences of collections previously assigned to H . crustuliniforme s.str. form a single monophyletic clade with bootstrap support of 92 % (ITS) and 100 % (MCM7, RPB2) in the single-gene analyses (see electronic supplementary material) and 96 % (Fig. 1b). In both Fig. 1a and b all collections assigned to ICG 5 by Aanen (1999) and Aanen and Kuyper (2004) are included in the H. crustuliniforme s.str. clade. As the bootstrap values of the H . crustuliniforme s.str. clade were smaller in ITS analyses than in analyses of other genes and on the whole, the branch length separating H . crustuliniforme s.str. from the rest of of H. crustuliniforme complex was rather small, we tested whether ITS-based distance values would support a distinction of H . crustuliniforme s.str. within the H. crustuliniforme complex. For this purpose,

Mycol Progress Fig. 1 a Best result of a ML analysis of ITS data of the Hebeloma crustuliniforme complex and closely related taxa. Bootstrap values were calculated from 650 replicates; b Best result of a ML analysis of ITS, RPB2 and MCM7 data of the H. crustuliniforme complex and closely related taxa. Bootstrap values were calculated from 700 replicates. A to C denote clades for which incompatible evolutionary histories are hypothesized in some single locus analyses (see running text and electronic supplementary material). Collection codes or Genbank acc. no. in black belong to the Hebeloma crustuliniforme complex. Collection codes in blue refer to collections that do not belong to the Hebeloma crustuliniforme complex based on morphology. Outgroup collections are in green (H. helodes sensu Aanen and Kuyper 2004). ITS-types from the same collection are denoted by ‘I’ and ‘II’ after the collection code. * epitype; ICG Intercompatibility group (from Aanen and Kuyper 1999)

a

AF124687 ICG9 AF124688 ICG9 HJB11328 Hebeloma HJB9819 helodes p.p. HJB8098 AF124681 ICG8 sensu Aanen 90 HJB12730 & Kuyper 2004 GC 07 09 29 03 WBS9508 ICG8 HJB10574 HJB11716 AF124694 ICG4 HJB10525II 82 80 HJB11687I HJB11687II AF124708 ICG3 JV08-451 HJB9267 HJB11100 HJB12007 HJB11051 AF124716 ICG2 AF124668 ICG1 AF124672 ICG1 HJB10384 JV96-341 PDD102994 HJB10282 AF124696 ICG2 HJB10446 AF124665 ICG1 HJB12655II AF124671 ICG1 HJB10766 HJB12655I HJB11107 97 HJB11079 AF124706 ICG7 HJB10525I HJB11133 90 HJB10427 AF124678 ICG14 HJB10422 JV08-278 HJB10260 AF124683 ICG5 89 HJB9053II HJB9053I HJB12264 WBS9581 ICG5 GL08704 WBS9689 ICG5 HJB11237 K(M)108529 JV00-643 HJB11215I Hebeloma HJB11215II crustuliniforme BR-MYCO 173990-69* s.str. HJB12842 HBo79139 MAK 08-10015 WBS9691 ICG5 JV91-670 WBS9546 ICG5 HJB9876 HJB13430 JV91-669 HJB12815 0.0020

the ‘intraspecific’ (i.e., within H. crustuliniforme s.str.) variation and the ‘interspecific’ variation were calculated, with ‘interspecific referring to the distance between ITS sequences belonging to the H. crustuliniforme s.str. clade and other sequences of the H. crustuliniforme complex. Using distance measure (1), 94 % of the H. crustuliniforme s.str. sequences show 1 bp or fewer differences in pairwise comparison [median(intraspecific)=0 bp, min(intraspecific)=0 bp, max(intraspecific)=2 b] and only 2 % of the interspecific pairwise comparisons result in 2 bp differences [median(interspecific)=4.5 bp, min(interspecific)=2 bp, max(interspecific)=11 bp]. When counting all gaps and not

b

84

0.0030

100 HJB11328 HJB9819 HJB8098 WBS9508 97 GC 07 09 29 03 HJB11728 HJB11716 HJB12730 98 HJB12007 98 HJB11079 HJB11107 100 HJB10282 HJB10446 C PDD102994 95 HJB10776 HJB12655 88 JV96-341 HJB10384 99HJB11100 B HJB11133 HJB11051 87 100 HJB10525 HJB9267 A JV08-451 HJB11687 99 HJB10260 99 HJB10422 100 HJB10427 JV08-278 GL08704 96 JV00-643 K(M)108529 HJB9053 WBS9689 ICG5 HJB12264 HJB11237 WBS9581 ICG5 HJB11215 WBS9691 ICG5 WBS9546 ICG5 BR-MYCO 173989-68* HJB9876 JV91-670 MAK 08-10015 HBo79139 HJB12842 HJB12815 HJB13430 JV91-669

Hebeloma helodes p.p. sensu Aanen & Kuyper 2004

Hebeloma crustuliniforme s.str.

equating ambiguous positions as with distance measure (2), the overlap of the intra and interspecific variation is more extensive, with only 60 % of the intraspecific pairwise comparisons resulting in 2 bp difference or less [median(intraspecific)= 2 bp, min(intraspecific)=0 bp, max(intraspecific)=12 bp] and the interspecific comparisons showing a minimum of 3 bp [median(interspecific)=9 bp, max(interspecific)=19 bp]. A sequence difference of 1 % roughly corresponds to 4.5 bp when only counting the spacer regions or to around 6.2 bp for the ITS1-5.8S rRNA-ITS2 span. The 5.8S rRNA sequence is identical in all cases.

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Discussion The main goal of the molecular analyses was to show the limits of Hebeloma crustuliniforme in the sense of the epitype. It is clearly a member of H . crustuliniforme in the sense of Aanen and Kuyper (2004), but monophyly and bootstrap support suggest that it can be further delimited to the clade called H . crustuliniforme s.str. in Fig. 1. Intragenomic variation does not indicate admixture or incomplete lineage sorting between H . crustuliniforme s.str. and the rest of the complex. The intragenomic ITS variation observed in two collections of H . crustuliniforme s.str. is contained within the clade. Furthermore, the mating test results of Aanen (1999) and Aanen and Kuyper (2004) strongly support the phylogenetic limits of the H. crustuliniforme s.str. clade as a biological species in that all sequenced members of ICG5 are contained in the clade, whereas all members of other ICGs are outside the clade. We therefore assume that H . crustuliniforme s.str. is the same as Aanen’s (1999) and Aanen and Kuyper’s (1999, 2004) ICG5. Aanen and Kuyper (2004) decided to include ICG5 in their concept of H. crustuliniforme together with four other ICGs in a multi-tiered decision process. At the time, only the sequence data of a single collection of ICG5 was available so that the molecular distinctness was not obvious. Intercompatibility group 5 was combined into H. crustuliniforme sensu Aaenen and Kuyper (2004) because it could not morphologically be unambiguously separated from a single other ICG (ICG1). The distinctiveness of H. crustuliniforme s.str. from the rest of the H. crustuliniforme complex has been shown in terms of monophyly and bootstrap, but the evolutionary distance is small. Automated identification of H. crustuliniforme s.str. based on ITS data may be challenging, even when using a 1 % cut-off value for species separation (Kõljalg et al. accepted, online), particularly when using the entire ITS PCR-fragment-length as 100 % reference figure. A distinct barcode gap, the quotient of the maximum of the intraspecific variation and the minimum of the interspecific variation only exists when looking at the median values of of intra and interspecific ITS variation. For the identification of a particular sequence, the distance measure employed may have a critical influence on the result. With distance measures like measure (2), capitalizing on the differences, the overlap between intra and interspecific sequence variation is larger. Even with a moderate measure like (1), ignoring gaps and stressing sequence similarity, a distinct barcode gap does not exist. However, when sequences are compared directly, as opposed to comparing the extremes of distance values, the recognition of H. crustuliniforme s.str. from ITS sequence data seems to be unambiguous: when blasting the H . crustuliniforme s.str. ITS sequences against our unpublished database, the best to at least fourth best results in terms of bits score are all H. crustuliniforme s.str. Critical sequences of the

rest of the H . crustuliniforme complex, i.e., sequences where the interspecific distance overlaps with the intraspecific distance of H. crustuliniforme s.str., are not erroneously identified as H . crustuliniforme s.str. in blast searches. Therefore, the identification of H . crustuliniforme s.str. may be less ambiguous than the fuzzy barcode gap suggests. Hebeloma crustuliniforme s.str. is described below as Hebeloma crustuliniforme (Bull.) Quél. emend. Vesterh., U. Eberh. & Beker. Figure 2 shows a map with the distribution of the European collections of H. crustuliniforme s.str. used in this paper, both for the species description and the molecular analyses (our analysis also includes two collections from separate locations on Long Island, NY in the USA). TAXONOMY Hebeloma crustuliniforme (Bull.) Quél. emend. Vesterh., U. Eberh. & Beker MycoBank MBT175887 (Figs. 3, 4 and 5) Typus : The holotype is plate 308 in Bulliard (1787). Unfortunately, as discussed earlier, it is not possible to unambiguously determine the precise species that Bulliard intended. Consequently, here we select an epitype. France: La Combe de la Serpente (N47.30240; E4.94631; alt. 434 m), on litter on calcareous soil in a conifer plantation, under Cedrus libanotica ssp.atlantica, 11 Oct. 2010, H. J. Beker and M. L. Beker (epitype BR BR-MYCO 173990–69, isoepitypes C C-F90146 and private collection Henry Beker HJB13713). Basidiomes usually in scattered groups, sometimes caespitose, sometimes solitary. Pileus up to 135 mm in diam., convex or umbonate, sometimes strongly umbonate; surface slightly viscid, tacky when moist never hygrophanous; cuticle colour from white to cream to buff, sometimes yellowish, but usually with at least some buff or brown in the centre, often almost unicoloured but usually becoming paler towards the margin; pileus margin usually straight, sometimes eroded in older fruitbodies. Lamellae emarginate, quite crowded (L=60–100) with a maximum depth of 4–9 mm; colour cream, alutaceous or brown when young, later tabacine following spore maturity; edge fimbriate, paler than gill surface; droplets on the lamella edge are usually present and visible to the naked eye; lamellules frequent. Stipe central, cylindrical more often clavate and sometimes subbulbous to bulbous, becoming hollow with age, 25–110 (115)×(6) 7–20 (26) mm and up to 27 mm at the base; white or alutaceous, with no visible discolouring when handled; surface dry, strongly floccose. Cortina not observed. Flesh rather thick, cream or pale brown. Smell raphanoid. Taste raphanoid, often bitter. Spore deposit brownish olive. Spores amygdaloid, with small apiculus and rounded at the end opposite the apiculus, with a distinct thinning of the spore wall but rarely with any papilla, guttulate with one or more oily drops, weakly ornamented to distinctly ornamented, sometimes with some sign of loosening perispore in a few spores and weakly dextrinoid ((O1)O2,O3; (P0)P1(P2);

Mycol Progress Fig. 2 Distribution map of the European collections of Hebeloma crustuliniforme as described in this paper (= H. crustuliniforme s.str.). The cross indicates the epitype locality

(D0)D1,D2); spore colour under the microscope pale brown to yellow brown; spore size based on n=61 spores of the epitype, 5 % to 95 % percentile range 10.5–12.6× 5.8– 6.5 μm, with median 11.3×6 .0 μm and av. 11.4×6.1 μm with S. D. length 0.60 μm and width 0.26 μm, Q value 5 % to 95 % percentile range 1.75–2.04, with median 1.87 and av. 1.88 with S. D. 0.09; spore size based on twenty collections 5 % to 95 % percentile range 9.6–13.5×5.5–7.2 μm, medians 10.7–12.1×6.0–6.7 μm and av. 10.8–12.2×6.1–6.7 μm with S. D. length 0.47–0.95 μm and width 0.22–0.41 μm, av. Q 1.70– 1.89. Basidia cylindrical to clavate and 4-spored, 26.5–40.2× 6.8–10.5 μm, with av. 27–37×7.4–9.8 μm. Pleurocystidia not found. Cheilocystidia clavate, clavate-stipitate and sometimes spathulate-stipitate, occasionally slightly swollen towards the base (clavate-lageniform) and occasionally with septa occasionally clamped, some median thickening, sometimes sinuate, never

Fig. 3 Collection BR-MYCO 173990–69 (epitype) of Hebeloma crustuliniforme, growing with oak. Photograph H. J. Beker

conglutinate; width of apex epitype 5 % to 95 % percentile range 5.6–7.3 μm, with median 6.5 μm and av. 6.5 μm with S.D. 0.57 μm; across twenty collections median 6.5–7.9 μm and av. 6.5–7.9. μm; with n at least 20 selected cheilocystidia of twenty collections the 5 % to 95 % percentile ranges are 29–86×4.9– 10.3×2.4–6.2×2.4–7.5 μm while the averages are 42–63×6.5– 7.9. × 3.4–4.7×3.5–5.0 μm and 58×6.5×3.4×3.5 μm av. for the epitype. The av. cheilocystidia ratios for the twenty collections were: A/M=1.61–2.32; A/B=1.52–2.32; B/M=0.90–1.22. Caulocystidia resemble cheilocystidia, but often rather more swollen towards the base, with more septa, up to 90 μm long and 10 μm wide at the apex. Pileipellis is an ixocutis with a relatively thick epicutis 150–350 μm, embedded hyphae up to 5–7 μm broad, smooth or sometimes encrusted, hyaline or occasionally pigmented. Subcutis yellowish and made up of

Fig. 4 Collection BR-MYCO 173990–69 (epitype) of Hebeloma crustuliniforme : a basidia×1,000, b spores×1,600, c cheilocystidia× 1,000. Scale bars=5 μm

Mycol Progress Fig. 5 Collection BR-MYCO 173990–69 (epitype) of Hebeloma crustuliniforme: a–b spores and spore ornamentation×1,600 in 5 % KOH; c–d spores and spore ornamentation×1,600 in Melzer’s reagent; e cheilocystidia×1,000; f cheilocystidia at gill edge×500; g basidium×1,000; h caulocystidia×1,000. Scale bars=10 μm. Photographs H.J. Beker

cylindrical to isodiametric elements. The trama below the subcutis contains cylindrical, spherical and thick sausage shaped elements up to 24 μm broad. Clamp connections present throughout the basidiome. Habitat H. crustuliniforme appears to be widespread across Europe. The description also includes two collections from the east coast of North America. It appears to grow in a variety of habitats including dunes, parklands and woodlands. Most records are from sandy or grassy soil, both acid and calcareous. We have no collections from arctic or alpine habitats. It appears to be mycorrhizally associated with a variety of trees. Records include Betula, Carpinus, Cedrus, Corylus, Picea, Pinus, Populus, Quercus and Tilia.

Additional specimens examined Channel Islands: Lavender (N49.19; W2.19; alt. approx. 15 m), on sandy, grassy soil with Betula sp., 11 Oct. 2003, S. Kelly (HJB9053); Les Quennevais, Les Blaches Barques (N49.2; W2.1; alt. approx. 50 m), on sandy soil in dune with Quercus ilex, 11 Nov. 1993, M. Rotheroe (K(M)108529; database record HJB9930). Cyprus: Troodos Mountains (N34.9137; E32.877; alt. approx.1410 m), on soil buried in pine needles with Pinus nigra, 10 October 2009, M. Loizides (HJB13430). Denmark: Århus, Universistetsparken (N56.167; E10.203; alt. approx. 12 m), on grassy soil with Quercus sp., 15 Oct. 1991, J. Vesterholt (C JV91-669; duplicate HJB12292); loc. cit. 15 Oct. 1991, J. Vesterholt (C JV91-670; database record HJB10832). England: London, (N51.4; E0.10; alt. approx. 20 m), on grassy

Mycol Progress

soil in urban garden with Quercus sp., 23 Nov. 2004, M. Tortelli (HJB11215); West Norfolk, Holkham Meals (N52.97098; E0.8057461; alt. approx. 0 m), on sandy soil in wooded dune with Pinus nigra and Salix sp., 28 Oct. 2003, C. Hobart (HJB9876). France: Haute Garonne, Toulouse University campus (N43.565; E1.461; alt. approx. 160 m), on grassy soil in parkland under Cedrus sp., 21 Nov. 2008, P. Jargeat, P.-A. Moreau (HJB12815). Germany: 156 Friederich-Engels-Allee, Wuppertal at hospital (N51.261;E7.171; alt. approx. 150 m), under Tilia sp ., 25 Sep. 1981, G. Zschieschang (GLM GL08704; database record HJB10977); Bad Kösen, SaxonyAnhalt (N51.13;11.72; alt. approx. 120 m),with Picea sp., 6 Oct. 2005, D. Penke (HBo79139;HJB12244). Macedonia: Kozhuf Mt; vicinity of Konjsko vill. Kamesha (N41.18913; E22.32317; alt. approx. 600 m), in broadleaf woodland on calcareous soil under Carpinus sp. and Quercus sp., 14 Oct. 2008, T. Tokov (MAK08-10015; duplicate HJB12851). Netherlands : Utrecht, Uithof (N52.086; E5.173; alt. approx. 0 m), with Tilia sp., 13 Oct. 1996, D. Aanen WBS9689; database record HJB12481); Gelderland, Rijswijk (N52.046; E4.33; alt. approx. 2 m), with Quercus sp., 20 Oct. 1996, D. Aanen (WBS9691; database record HJB12803); Utrecht (N52.00536; E5.0710; alt. approx. 10 m), with Tilia sp., 5 Nov. 1995, D. Aanen (WBS9581; database record HJB12807); Drenthe, Roden (N53.0818; E6.25569; alt. approx. 2 m), with Corylus avellana and Quercus sp., 5 Oct. 1995, D. Aanen (WBS9546; database record HJB12811). Portugal: Minho, Ponte de Barca (N41.81; W8.42; alt. approx. 35 m), under Populus sp.,11 Nov. 2000, P. Boisen Hansen, J. Vesterholt (C JV00-643; duplicate HJB10902). Spain : Madrid, Miraflores (N40.8699833; W3.7657; alt. approx. 1517 m), on grassy soil in mixed woodland with Pinus sp. and Populus sp., 10 Nov. 2004, H. Beker (HJB11237). United States: Brookhaven State Park, Long Island (N40.9218640; W72.8661350; alt. approx. 21 m) with Pinus rigida and Quercus sp ., 10 Oct. 2008, J. Horman (HJB12842); Edgewood National Reserve, Long Island (N40.78325; W73.3049833; alt. approx. 43 m) on soil in litter with Pinus rigida and Quercus sp., 17 Nov. 2007, H. Beker (HJB12264). Comment The taxon forms part of the complex that is often referred to as the H. crustuliniforme complex and was described as H. crustuliniforme by Aanen and Kuyper (2004). Characteristically, members of H. crustuliniforme in the wider sense have medium to large fruitbodies with a distinctly floccose stipe. Hebeloma crustuliniforme as described here, i.e. H. crustuliniforme s.str. in Fig. 1, has the characteristic cheilocystidia of section Denudata, swollen at the apex and then tapering, cylindrical or slightly swollen towards the base (predominantly clavate-stipitate or spathulate-stipitate), but probably with the smallest apex within the H. crustuliniforme complex with an average rarely above 7.4 μm and always below 8 μm. The spores of H. crustuliniforme as described

here are quite long for this complex, the average spore length is rarely below 10.8 μm, and the average spore width always appears to be above 6 μm. These characters are usually sufficient to distinguish it from the other medium to large taxa within the H. crustuliniforme complex. Whereas morphological identification of H. crustuliniforme might be ambiguous in rare cases where the spores of some other members of the H . crustuliniforme complex are large or the spores of H . crustuliniforme are small and the cheilocystidia apex measurements might also be ambiguous, single locus molecular data from any of the loci used here is reliable and sufficient for species identification, regardless whether testing for monophyly or doing blast searches. This assessment is based almost exclusively on data of European collections. The description of H . crustuliniforme given here fits well with the description of ICG 5 given by Aanen (1999) apart that his cheilocystidia measurements are slightly larger. This is very likely due to following different selection strategies when measuring cheilocystidia. Among the H . crustuliniforme complex, the taxon described here probably has the darkest coloured pileus and is often a uniform buff. Thus we believe it most closely approaches the plate (iconotype) of Bulliard and thus we have chosen the epitype for Hebeloma crustuliniforme from this taxon.

Acknowledgments We are very much obliged to L. Gosling who made the distribution map for this and previous publications and to G. Walther for providing the microscopic line drawings. The authors would like to thank the herbaria in C (Copenhagen), GLM (Görlitz), IB (Innsbruck), K (Kew), L (Leiden), LY (Lyon), PDD (Auckland) for the loan of collections for study and sequencing, T. Kuyper for making material available from D. Aanen’s collections from WBS (Biologisch Station Wijster, Wageningen University) and BR (Meise) for kindly managing the deposits. Furthermore, we very much appreciate the help of M.L. Beker, P. Boisen Hansen, G. Corriol, C. Hobart, J. Horman, S. Kelly, P. Leonard, M. Loizides, P.-A. Moreau, P. Jargeat, T. Tokov and an anonymous collector for supplying us with interesting and exciting Hebeloma collections. We would also thank P. Kirk for providing us with statistics from the Fungal Records Database of Britain and Ireland. Numerous people have helped in the lab to generate Hebeloma sequence data that were used directly or indirectly in this study. We would like to thank K. Dukik, U. Fürst, R. Gadjieva, S. Garnica, M. Jonsson, C. Lundström, J. Petterson, D. Öncü and J. Schade as well as the Uppsala University Genome Center and the Hubrecht Institute sequence facility.

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