MycoKeys 90: | 63-202 (2022) sae A peer-reviewed open-access journal doi: 10.3897/mycokeys.90.85267 RESEARCH ARTICLE . 03 MycoKkeys https://mycokeys.pensoft. net Launched to accelerate biodiversity research Not (only) poison pies -— Hebeloma (Agaricales, Hymenogastraceae) in Mexico Ursula Eberhardt', Alejandro Kong’, Adriana Montoya’, Nicole Schiitz', Peter Bartlett?, Henry J. Beker*>* I Staatliches Museum fir Naturkunde Stuttgart, Rosenstein 1, 70191 Stuttgart, Germany 2 Centro de Inves- tigacion en Ciencias Biolégicas, Universidad Auténoma de Tlaxcala, Km 10.5 carretera San Martin Texmelu- can-Tlaxcala, San Felipe Ixtacuixtla, Tlaxcala, 90120, Mexico 3 La Baraka, Gorse Hill Road, Virginia Water, Surrey GU25 4AP United Kingdom 4 Rue Pere de Deken 19, B-1040 Bruxelles, Belgium § Royal Holloway College, University of London, Egham, UK 6 Plantentuin Meise, Nieuwelaan 38, B-1860 Meise, Belgium Corresponding author: Ursula Eberhardt (ursula.eberhardt@smns-bw.de) Academic editor: Maria-Alice Neves | Received 12 April 2022 | Accepted 7 June 2022 | Published 30 June 2022 Citation: Eberhardt U, Kong A, Montoya A, Schiitz N, Bartlett PR. Beker HJ (2022) Not (only) poison pies — Hebeloma (Agaricales, Hymenogastraceae) in Mexico. MycoKeys 90: 163-202. https://doi.org/10.3897/mycokeys.90.85267 Abstract The species of Hebeloma have been little studied in Mexico, but have received attention as edibles and in trials to enhance production of edible fungi and tree growth through inoculation of seedlings with ecto- mycorrhizal fungi. Here we describe three new species of Hebeloma that are currently known only from Mexico. These species belong to separate sections of the genus: H. ambustiterranum is a member of H. sect. Hebeloma, H. cohaerens belongs to H. sect. Theobromina, while H. magnicystidiatum belongs to H. sect. Denudata. All three species were collected from subtropical pine-oak woodland; all records of H. cohaerens came from altitudes above 2500 m. Hebeloma ambustiterranum is commonly sold in the local markets of Tlaxcala as a prized edible mushroom. An additional nine species are reported from Mexico, of which eight are new records for the country: H. aanenii, H. eburneum, H. excedens, H. ingratum, H. neurophyl- lum, H. sordidulum, H. subaustrale and H. velutipes. First modern descriptions of H. neurophyllum and H. subaustrale, originally described from the USA, are given here. Keywords barcodes, Basidiomycota, ectomycorrhizal fungi, edible fungi, 3 new species, type studies Copyright Ursula Eberhardt et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 164 Ursula Eberhardt et al. / MycoKeys 90: 163-202 (2022) Introduction Arguably, the best recognized vernacular English name for the genus Hebeloma is poi- son pie, although this name is often reserved for H. crustuliniforme, and other species within the genus are qualified versions of this name, e.g. H. mesophaeum is the veiled poison pie and H. pusillum is the dwarf poison pie (https://www.britmycolsoc.org.uk/ library/english-names, accessed 18 Nov 2021). The name poison pie suggests what is, certainly in Europe, believed to be true for all members of the genus: that they are poisonous, or even if they were not, all too easily mixed up with poisonous members of the genus. Collecting Hebeloma for human consumption is generally discouraged (Bresinsky and Besl 1990; Benjamin 1995). In Mexico, the main interest in Hebeloma from the local community was either in the context of edibility (e.g., Montoya et al. 2008; Reyes-Lopez et al. 2020) or with re- gard to the inoculation of trees of forest importance with ectomycorrhizal fungi (Pérez- Moreno et al. 2020 and references therein; Pérez-Moreno et al. 2021). A number of Hebeloma species were mentioned in these articles, including H. alpinum, H. helodes, FH. leucosarx and H. mesophaeum. We have not had the opportunity to examine the material used in the respective publications. Given the difficulty surrounding species concepts of this genus, the pres- ence of these species in Mexico should be treated with caution. Both, with regard to the consumption of mushrooms and the inoculation of tree seedlings, it would be ad- vantageous to have a clear understanding of the species involved and the morphological and molecular characters that define them to recognize or verify collections or strains. To the best of our knowledge, Hebeloma are not included in commercial ectomy- corrhizal fungi mixtures currently sold to enhance tree growth, but it is one of the few genera that have been used in numerous nursery trials and transplanting experiments (e.g., Castellano and Molina 1989; Barroetavefia and Rajchenberg 2005; Gagné et al. 2006; Oliveira et al. 2010 and see below). Owing to the difficulties delimiting and identifying Hebeloma species, members of the genus have often been treated as if they all shared the same ecological traits. This is clearly not the case (Beker et al. 2016). From the taxonomic side, the Hebeloma of North America have been largely ne- elected since the work of Hesler and Smith in the 1970s and 1980s (Hesler 1977 and his unpublished manuscript on North American species of Hebeloma, Smith et al. 1983) and never extensively studied within Mexico. This lack of understanding of spe- cies concepts can be illustrated by reference to observation websites. For example, iN- aturalist (https://www.inaturalist.org/observations?place_id=6793&taxon_id=192716 accessed on 12 March 2021) listed 41 Hebeloma observations for Mexico, but just six of these observations had species names attached: one was referred to H. mesophaeum and five were referred to H. crustuliniforme. Mushroom Observer (https://mushroo- mobserver.org/observer/advanced_search?q=leMh6 accessed 12 March 2021) listed just eleven Hebeloma records from Mexico, none of which were identified to spe- cies level. The Global Biodiversity Information Facility GBIEorg (GBIF Occurrence Download https://doi.org/10.15468/dl.wd7f75 accessed 18 November 2021) gave Hebeloma in Mexico 165 169 results for Hebeloma from Mexico, of which 60 were identified to species level: H. alpinum (1), 1. crustuliniforme (17), H. edurum (1), H. fastibile (19), H. mesophaeum (16), H. sacchariolens (1) and H. sinapizans (5). MycoPortal (https://mycoportal.org/ portal/collections/list.php accessed on 18 November 2021) gave 105 results for Hebe- loma of Mexico. 100 of these records were from the National Herbarium of Mexico Fungal Collection (MEXU), four were from the Field Museum of Natural History (F) and one was from USDA, the United States National Fungus Collections (BPI). Of these 105 collections, 86 had no species name given, ten were identified as H. fasti- bile, five as H. sinapizans, three as H. mesophaeum and one as H. sacchariolens. There were, of course, overlaps between these databases, and one should be cautious of deter- minations given the historical confusion regarding species definitions, but all species records indicate just 6 species recorded: H. alpinum, H. crustuliniforme, H. laterinum (= H. edurum, H. fastibile), H. mesophaeum, H. sacchariolens and H. sinapizans. Beker et al. (2016) published a monograph on Hebeloma of Europe to provide a new foundation for the understanding of species of this genus, on which future studies could be built. Although this monograph only addressed the genus within Europe, it has provided a base both morphologically and molecularly. Since the publication of that monograph, a number of papers have been published describing new species of Hebeloma as well as resurrecting long forgotten names that can now be confirmed as valid (e.g., Cripps et al. 2019; Eberhardt et al. 2020a, 2020b, 2021a, 2021b, 2022a, 2022b; Monedero and Alvarado 2020). Within this paper, we present a list of Hebeloma species we have found during analysis of herbarium collections from Universidad Auténoma de Tlaxcala (TLXM). The 90 collections studied came from two principal areas in Chihuahua and Tlax- cala but also included a few collections from the regions of Mexico City and Puebla. Within this set, two species were rediscovered, H. neurophyllum (Atkinson 1909) and H. subaustrale (Murrill 1945), originally described from the US. The identifications were verified by morphological and molecular type studies. Three species new to sci- ence were discovered and are described below as H. ambustiterranum, H. cohaerens and H. magnicystidiatum. These species belong to separate sections of the genus and are described below. Materials and methods All the material studied were dried specimens from the Universidad Autdnoma de Tlaxcala (TLXM). The collections sites are shown in Fig. 1. These collections were compared to material collected for the Hebeloma project (Beker et al. 2016). Coor- dinates were obtained in the field by GPS or were approximated from the collection data. Approximations of elevations (m above sea level), where not recorded at time of collection, were deduced using Google Earth (Google Earth Pro Version 7.3.4.8248). Sequences were obtained from the dried basidiomes by direct sequencing. At least the ITS (barcode) locus was generated for all Mexican collections and, in a number 166 Ursula Eberhardt et al. / MycoKeys 90: 163-202 (2022) ia! gi Mexico State @laxcala® Onc. Puebla Hebeloma ambustiterranum Hebeloma cohaerens Hebeloma magnicystidiatum Hebeloma neurophyllum Hebeloma subaustrale other Hebeloma spp. 1000 km Figure |. Collection sites of studied material. Scale bar 1000 km. The map was generated with QGIS version 3.16.15 using WGS84, EPDG: 4326 (QGIS Association, QGIS.org, 2022). Shapefiles were pro- vided by the Database of Global Administrative Areas (GADM); Accessed April 2018 to March 2022. of cases, additional loci were sequenced. Internal transcribed spacer sequences were generated following methods detailed in Eberhardt (2012) and Cripps et al. (2019); MCM7 (minichromosome maintenance complex component 7, partial) data follow- ing Eberhardt et al. (2016a); RPB2 and TEFIa sequences following Eberhardt et al. (2021a); and sequences of two variable regions (V6 and V9) of the mitochondrial SSU followed Gonzalez and Labarére (1998). Sequencing was carried out by LGC Genom- ics (Berlin, Germany). Sequences were edited using Sequencher vs. 4.9 (Gene Codes Corp., Ann Arbor, Michigan). Newly generated sequences were accessioned to Gen- Bank (ON167764—ON167898, ON168958—ON 168966, ON202494—ON202614 and ON237944—-ON237985), Suppl. material 1: Table S1 summarizes all sequences used in the analyses, including those previously published in the context of a number of publications (Eberhardt et al. 2009, 2013, 2015, 2016a, 2016b, 2021a, 2022a, 2022b; Eberhardt and Beker 2010; Beker et al. 2010, 2013, 2016; Schoch et al. 2012; Cripps et al. 2019). Sequence alignments were done online in MAFFT using the E-INS-i option (Katoh et al. 2005, 2019) or locally with the “Mafft-globalpair” setting of MAFFT 7.471 (Ka- toh and Standley 2013). Alignments were done, viewed and reformatted in ALIVIEW 1.27 (Larsson 2014). Phylogenetic analyses (ML) were run in IQ-TREE (Nguyen et al. 2015) online (Trifinopoulos et al. 2016). Model selection (Kalyaanamoorthy et al. 2017) was done using the BIC criterion, including FreeRate models and merging partitions if possible (protein coding loci were originally partitioned according to position, cod- ing and non-coding). Branch support was obtained through 1000 replicates of ultrafast Hebeloma in Mexico 167 bootstrap (ufb; Minh et al. 2013; Hoang et al. 2018) and SH-like approximate likeli- hood ratio tests (SH-aLRT; Guindon et al. 2010). Support values are given as (SH-aLRT [%]/ufb [%]), for SH-aLRT support = 85% and ufb support = 95%. Nexus files with alignments and trees, including all single locus trees, are available as Suppl. material 2. Alignments were made for sections including new or rediscovered species, i.e., for H. sect. Hebeloma, H. sect. Naviculospora, H. sect. Theobromina and H. sect. Velutipes, including loci that were known to facilitate species recognition in the respective section (Beker et al. 2016). Sequences of types were included if available unless missing data (short sequences) had an adverse effect on the taxonomic resolution of the result. The selection of loci, additional species and taxa used for rooting was guided by previous results (Beker et al. 2016; Cripps et al. 2019; Eberhardt et al. 2021a, 2021b, 2022a, 2022b) — and by the loci that could be generated from the collections available. Prior to concatenation, single locus trees (see Suppl. material 2) were generated. Conflicts were detected using the principle by Kauff and Lutzoni (2002), assuming a conflict to be significant if two different relationships for the same set of taxa, one being monophy- letic and the other non-monophyletic, were supported by SH-aLRT support > 85% or ufb support = 95%. Alignments of different loci were concatenated and analyzed, indicating branches with conflicting results from single locus analyses by dashed lines. Distances between sequences were calculated from the alignments used for the ML analyses as p-distances with pairwise deletion of gaps in MegaX (Kumar et al. 2018; Stecher et al. 2020). The UNITE database (Koljalg et al. 2013, 2020) and plutof (Ab- arenkov et al. 2010) were used for sequence searches, directly and via BLAST and for matching sequences to SH (species hypotheses). Details of morphological analyses were provided in Beker et al. (2016). The amount of macroscopic detail available to us varied hugely from collection to collection as it was dependent on the detail provided by the collector. For recent collections where one of the authors was the collector, each specimen was photographed and observed both in the field when characters were still fresh, and later in the laboratory. Fresh basidi- omes of each specimen were dried using a food dehydrator. All microscopic analysis was carried out on dried material, using a Leica DMRZA2 microscope with a Leica DFC495 camera connected to a computer running Leica Ap- plication Suite (LAS) V4 software. The basidiospores were first studied in Melzer’s reagent to assess the shape, degree of dextrinoidity, ornamentation and the degree of loosening of the perispore. For the assessment of the degrees of ornamentation (O0, O1, O2, O03, O4), of the loosening perispore (PO, P1, P2, P3) and for the dextrinoidity (DO, D1, D2, D3, D4), we used Beker et al. (2016) and Vesterholt (2005). A number of photographs were taken of the basidiospores at x500 and x1600, which were then measured using the LAS soft- ware. For each collection, wherever possible, at least 50 basidiospores were measured in Melzer’s reagent, excluding the apiculus. As discussed in Beker et al. (2016), the difference in Hebeloma basidiospore size from dried material, measured in Melzer’s reagent and 5% KOH, is negligible. The maximum length and width of each spore was measured, and its Q value (ratio of length to width) calculated. Average length, width, 168 Ursula Eberhardt et al. / MycoKeys 90: 163-202 (2022) and Q value were calculated and recorded alongside the median, standard deviation, and 5% and 95% percentiles. The material was then examined in 5% KOH. Photographs were taken of the ba- sidiospores and also of the cheilocystidia (and pleurocystidia if any were present) and basidia at x500 and x1000. Because of the complex shapes of the cheilocystidia four measurements were made: length, width at apex (A), width at narrowest point in cen- tral region (M), and maximum width in lower half (B). The measurements were given in this order, and an average value was calculated for each of these measurements. The average width of the cheilocystidium in the vicinity of the apex appears to be an impor- tant character in the separation of species within Hebeloma (Vesterholt 2005). It is also important, when determining this average width near the apex, not to be selective with regard to the cystidia chosen for measurement. To determine the average width at the apex, about 100 cheilocystidia were measured on the lamella edge. For other measure- ments, some 20 cheilocystidia, separated from the lamella edge, were measured from each collection. For each cheilocystidium the ratios A/M, A/B, and B/M were calcu- lated and averaged across all cheilocystidia measured. For all other details with regard to our methodology, see Beker et al. (2016). Each collection studied has a database record number associated with that col- lection (beginning HJB); we give these numbers as we intend to make the database publicly available. If no other herbarium abbreviation or herbarium accession number is given, the HJB number is also the collection number within H.J. Beker’s herbarium. Species were identified considering morphological and molecular data. In cases in which molecular data were not conclusive (as e.g., for H. eburneum and H. velutipes, or could not be obtained, as for the type of H. subaustrale), species identification followed morphology. For species not discussed in detail here, please refer to species descriptions in Beker et al. (2016) and Eberhardt et al. (2021a, 2022a). Results It appears that all of the species found in our sample, other than Hebeloma mesophae- um, ate new species records for Mexico. Fig. 1 shows the distribution of these fungal collections in Mexico; Suppl. material 1: Table S1 lists all collections utilized during this study, including those not specifically discussed in the Taxonomy part. The analysis of taxa from H. sect. Hebeloma (from Mexico H. ambustiterraneum, H., excedens and H. mesophaeum) included ITS, RPB2 and Tefla data, and 67 collec- tions from 13 species. Hebeloma sordescens (H. sect. Hebeloma) was used for rooting. Hebeloma ambustiterranum was monophyletic in all single locus results and received support in ITS (100/100%) and RPB2 (85/98%). Conflicts between ITS and the other two loci were observed in relation to the position of H. pubescens (p.p.) and H. subtor- tum (ITS with H. excedens, H. mesophaeum and H. psammophilum; RPB2 and TEF 1a with H. colvinii and H. velatum [= H. dunense, Eberhardt et al. 2022a] and within H. pubescens {collection HJB12057]). Neither of these conflicts were considered rel- Hebeloma in Mexico 169 evant in the current context. The concatenated alignment spanned 2205 positions. The clade of H. ambustiterranum (Fig. 2) received full (100/100%) support. This result sup- ported morphology in that H. ambustiterranum is a good species new to science. Neither H. excedens nor H. mesophaeum were resolved (Fig. 2); the Mexican collections of these two species were placed among other members of H. excedens and H. mesophaeum. The analysis for H. sect. Denudata (in Mexico H. aanenii, H. eburneum, H. ingra- tum, H. magnicystidiatum and H. sordidulum) was based on ITS, mitSSU V6 and V9 of 78 collections from 17 species. Hebeloma echinosporum and H. populinum (H. sect. Denudata, subsect. Echinospora) were used for rooting. In the ITS tree, H. magnicystidi- atum was part of the H. sordidulum clade (90/—%), which was included in a weakly supported clade (90/—%) with all other members of H. subsect. Clepsydroida consid- ered in the analysis. Neither of the mitSSU results contradicted this relationship with any support, but there were conflicts between the ITS and mitSSU results and between the two mitSSU results in relation to the limits of the subsections and the relationship of H. hiemale (H. subsect. Hiemalia) and H. subsect. Clepsydroida and H. subsect. Crustuliniformia. In spite of this, the alignments were concatenated. The resulting phy- logenetic hypothesis (Fig. 3) showed H. magnicystidiatum outside the clade of H. sor- didulum (which was only weakly supported, 85/—%), but on a relatively long branch, thus supporting morphology that H. magnicystidiatum is a separate species. Because of existing conflicts, molecular data could not resolve the position of H. magnicystidiatum in any of H. subsects. Clepsydroida, Crustuliniformia or Hiemalia. The Mexican collections of H. aanenii clustered with their conspecifics from other countries, while the Mexican collections of H. eburneum were not in the same clade as H. eburneum collections from other countries, both clades received some support, one by ufb, the other by SH-aLRT (see Fig. 3). The only single locus tree showing a Mexican H. eburneum clade is mitSSU V6 (86/95% support). Both H. eburneum clades were, as well as H. aanenii, in what Beker et al. (2016) termed the H. alpinum- complex (94/97% support). The Mexican collection of 1. ingratum was included in the 1. ingratum clade (93/98% support); the Mexican collection of H. sordidulum was included in the respective species clade, which only received 87/— support. The analysis for H. sect. Velutipes (in Mexico H. neurophyllum and H. velutipes) was based on ITS, RPB2, TEF1a and mitSSU V6 of 59 collections from 12 species. Hebeloma bulbiferum and H. sinapizans (H. sect. Sinapizantia) were used for rooting. Hebeloma neurophyllum received good support (95/95%) in the ITS result, and is paraphyletic in relation to H. erebium in the RPB2 and TEF/a results, and in relation to H. celatum in the mitSSU V6 result. In spite of a number of conflicts concerning interspecific relationships within H. sect. Velutipes—intraspecific conflicts were not detected—the different single locus alignments were concatenated. ‘The alignment included 2670 positions. In the anal- ysis of the concatenated dataset (Fig. 4), H. neurophyllum was well supported (97/99%), as were H. celatum (97/ 99%) and H. erebium (98/100%). Thus, molecular data as well as morphological characters (see below) supported H. neurophyllum as a good species. Hebeloma velutipes was paraphyletic in relation to the other member species of the H.. velutipes complex clade (H. incarnatulum, H. leucosarx and H. subconcolor). The 170 Ursula Eberhardt et al. / MycoKeys 90: 163-202 (2022) [100/100] [100/100] 100/99 Hebeloma marginatulum & H. pascuense* H. alpinicola HJB12439 US H. alpinicola C-F-101621 HJB15611 GE H. alpinicola MICH 5549 HJB1000311 US* H. alpinicola HJB11019 IS H. mesophaeum HJB10683 UK H. excedens TLXM HJB16817 MX H. mesophaeum HJB11944 SJ H. mesophaeum HJB12114 FR H. mesophaeum C JV-03-547 HJB10854 DK* H. excedens NYS-F-001123 HJB1000268 US* H. mesophaeum SWGC GM14B-025 HJB14858 CA H. mesophaeum TLXM HJB16737 MX H. mesophaeum HJB10166 BE H. excedens HJB16980 US H. excedens AG20846 HJB15702 US H. excedens TLXM HJB16734 MX H. mesophaeum HJB11050 IS H. mesophaeum C-F-104301 HJB17068 GL 99/100 Hebeloma psammophilum* TLXM HJB16803 MX TLXM 6155 MX* TLXM HJB16753Mx 4 Mebeloma TLXM HJB16805 Mx | @Mbustiterranum* TLXM HJB16750 MX sp. nov. TLXM HJB16748 MX TLXM HJB16799 MX 100/100 99/100 Hebeloma subtortum* 90! = —<< | Hebeloma pubescens* 700 Hebeloma colvinii* ;_—_ ee __o ! . 0.004 << | Hebeloma velatum Figure 2. ML topology of concatenated ITS, RPB2 and TEF1a sequences of Hebeloma sect. Hebeloma. | | ! ! ! | ! ! ! | I ! ! | | ! ! | | ! ! ! L Branch support was obtained through 1000 replicates of SH-like approximate likelihood ratio tests and ul- trafast bootstrap annotated SH-aLRT/utb at the branches for > 85% SH-aLRT and = 95% for ufb support. Dotted lines indicate parts of the tree where conflicts between single locus results were observed. Hebeloma sordescens (H. sect. Hebeloma) was used for rooting. Collections indicated with * are types; clade names indicated by * include type sequences. Collections and species names in red refer to Mexican material. Hebeloma in Mexico 171 I< | Hebeloma echinosporum* & H. populinum [100/100] sea | Hebeloma hiemale* 99/100 Hebeloma crustuliniforme* H. eburneum HJB10290 BE H. eburneum HJB12670 PL H. eburneum DUKE 0351128 HJB17317 US H. eburneum C-F-111117 HJB18936 GL H. aanenii PDD 102994 HJB10692 NZ H. aanenii BR-MYCO 173987-66 HJB12630 PL* H. aanenii TLXM HJB16741 MX H. aanenii HJB10435 CA H. aanenii TLXM HJB16745 MX H. aanenii TLXM HJB16740 MX H. aanenii TLXM HJB16742 MX H. alpinum CLC2855 HJB15331 US H. alpinum H. alpinum HJB12005 SV complex H. alpinum HJB11132 HE H. geminatum TURA 17928F HJB10961 EE H. geminatum C C-F-90152 HJB10833 DK* H. geminatum HJB11801 FR TLXM HJB16796 MX TLXM HJB16760 MX TLXM HJB16775 MX TLXM HJB16758 MX Hepelomercouineuin TLXM HJB16761 MX p.p. ‘Mexico TLXM HJB16786 MX TLXM HJB16792 MX LY BR64-24 HJB1000040 FR* [100/100] HJB16994 US Hebeloma ingratum* HJB11311 BE L WAG-W9573 HJB12534 NL Hebeloma cinnamommeum* 100/100 Hebeloma limbatum* 99/100 Hebeloma ammophilum HJB12287 US HJB16978 US TLXM HJB16754 MX Hebeloma sordidulum DPL11915 HJB15700 US MO231249 HJB15800 CA 86/- | HJB12269 US H. magnicystidiatum sp. nov. TLXM 6157 MX* 100/100 4 Hebeloma matritense* 1 99/99 WOE Hebeloma cavipes & H. vaccinum Figure 3. ML topology of concatenated ITS, mitSSU V6 and V9 sequences of Hebeloma sect. Denudata. Branch support was obtained through 1000 replicates of SH-like approximate likelihood ratio tests and ultrafast bootstrap annotated SH-aLRT/ufb at the branches for > 85% SH-aLRT and = 95% for ufb sup- port or by thick lines in the case that at least one of the support values is equal to or exceeds the limits. Dotted lines indicate parts of the tree where conflicts between single locus results were observed. Hebeloma echinosporum and H. populinum (H. subsect. Echinospora of H. sect. Denudata) were used for rooting. Collections indicated with * are types; clade names indicated by * include type sequences. Collections in red refer to Mexican material. 2. Ursula Eberhardt et al. / MycoKeys 90: 163-202 (2022) position of the Mexican collections of H. velutipes in a separate clade (97/99%) was only supported by the mitSSU V6 data. The analysis for H. sect. Theobromina (in Mexico H. cohaerens) was based on ITS, MCM7 and RPB2 of 32 collections from nine species. Hebeloma sinapizans was used for rooting. Hebeloma cohaerens was supported by all three single locus analyses (96- 97/95—100%) and received full (100/100%) support in the analysis of the concatenat- ed data (2152 bp) (Fig. 5A). No conflicts were found between the single locus results. Thus, both molecular results and morphology supported H. cohaerens as a new species. The analysis for H. sect. Naviculospora (in Mexico H. subaustrale) was based on the ITS of 24 collections of eight species and included 703 positions. Hebeloma islandicum, provisionally placed by Beker et al. (2016) in H. sect. Naviculospora to avoid creating a monospecific section for the species, was used for rooting. Holotype sequences generated by PB. Matheny and A.D. Wolfenbarger of H. angustisporium (NR_119890, Schoch et al. 2014) and of H. perangustisporium (HQ179680, unpublished, submitted 23 Aug 2010) and by H. Gordon of H. pungens (MW412387, unpublished, submitted 28 Dec 2020) were identical or almost identical with our sequences but had shorter read length in the ana- lyzed region. Thus, only the sequences generated by us were considered in the analysis. The holotype sequences of H. angustisporium and H. perangustisporium, as well as three mor- phologically matching collections formed a clade supported by 97/97% among all other recognized members of H. sect. Naviculospora. Morphologically, the H. angustisporium and H. perangustisporium agree with H. subaustrale, which is the oldest of the three names. Thus, although no sequence data could be obtained for the type of H. subaustrale, the clade is referred to as 1. subaustrale in Fig. 5B, and H. subaustrale is accepted and described below. Taxonomy For species described from Europe please refer to Beker et al. (2016); for H. excedens and H. sordidulum to Eberhardt et al. (2022a) and for H. excedens also to Cripps et al. (2019). Hebeloma ambustiterranum A. Kong & Beker, sp. nov. MycoBank No: 842826 Figs 6—7 Type. Mexico. Tlaxcala: La Malinche National Park, 19.2749°N, 97.9825°W, alt. approx. 2800 m, on burnt soil in coniferous woodland under Pinus montezumae and P teocote, 8 Jul 2017, H.J. Beker HJB16802 (holotype TLXM 6155; isotype BR 5020224874626V); GenBank ITS ON202501. Diagnosis. ‘The small ellipsoid, non-dextrinoid, almost smooth basidiospores (on average 8.0—10.2 x 5.6—-6.5 um) and at least 50 full length lamellae distinguish this species from all other known North American Hebeloma species and the ITS sequence differentiates this species from all other known species, worldwide. Hebeloma in Mexico 173 Hebeloma bulbiferum & H. sinapizans* 100/100 Hebeloma aestivale 98/100 Hebeloma erebium* TLXM AK3782 MX DENA-61424 HJB17897 US 97/99| | 41JB18101 CA DPL11907 HJB15699 US HJB16991 US HJB17981 CA WTU-F-039596 HJB1000558 US* Hebeloma neurophyllum* 100/100 Hebeloma albidulum 93/100 (alee Hebeloma incarnatulum* H. velutipes HJB11315 PT H. velutipes C-F-44818 HJB10329 IT H. velutipes HJB11756 FR H. velutipes HJB11951 NO H. velutipes HJB10588 UK H. velutipes HJB10350 BE Hebeloma subconcolor* Hebeloma leucosarx* H. velutipes C-F-103582 HJB16689 GL H. velutipes NS3095 HJB18774 US TLXM AK3749 HJB16797 MX TLXM HJB16815 MX H., velutipes TLXM HJB16816 MX p.p. ‘Mexico’ 0.02 TLXM HJB16738 MX Figure 4. ML topology of concatenated ITS, RPB2 and TEFla and mitSSU V6 sequences of Hebe- loma sect. Velutipes. Branch support was obtained through 1000 replicates of SH-like approximate likeli- hood ratio tests and ultrafast bootstrap annotated SH-aLRT/ufb at the branches for > 85% SH-aLRT and > 95% for ufb support or by thick lines in the case that at least one of the support values is equal to or exceeds the limits. Dotted lines indicate parts of the tree where conflicts between single locus results were observed. Hebeloma bulbiferum and H. sinapizans (H. sect. Sinapizantia) were used for rooting. Col- lections indicated with * are types; clade names indicated by * include type sequences. Collections in red refer to Mexican material. 174 Ursula Eberhardt et al. / MycoKeys 90: 163-202 (2022) A + Hebeloma sinapizans [100/100] 100/100 Hebeloma parvicystidiatum* 99/100 100/100 Hebeloma alboerumpens* 100/100 Hebeloma plesiocistum* [100/100] 100/100 _/! Hebeloma vesterholtii* 100/100 100/100! Hebeloma theobrominum* TLXM 6156 MX* TLXM GF1866 HJB16779 MX H. cohaerens* TLXM AME3101 HJB17732 MX § SP. Nov. TLXM AK-17-08HJB17737 MX 96/98] _100/100 94/99 ; ; 100/100 Hebeloma griseopruinatum Hebeloma erumpens 0.007 B i Hebeloma islandicum* [100/ 100] H. naviculosporum AH14256 HJB1000023 ES* H. naviculosporum LB11081701 HJB14211 ES H. naviculosporum HJB13807 DE H. naviculosporum KRAM-F-57436 HJB14568 SK TLXM HC1155 HJB16793 MX DBSPFS -2011-63 HJB17796 US [100/ H. angustisporium TENN-F-023364 HJB1000314 US* 100] HJB18418 US H. perangustisporium TENN-F-013890 HJB1000450 US* Hebeloma subaustrale Hebeloma nanum* Hebeloma pungens HJB1000474 US* 100/100 Hebeloma catalaunicum* 7 Hebeloma avellaneum* 0.003 Figure 5. ML topologies with branch support obtained through 1000 replicates of SH-like approximate likelihood ratio tests and ultrafast bootstrap annotated SH-aLRT/utb at the branches for > 85% SH-aLRT and > 95% for ufb support. Collections indicated with * are types; clade names indicated by * include type sequences. Collections in red refer to Mexican material A concatenated ITS, MCM7 and RPB2 sequences of Hebeloma sect. Theobromina, rooted with H. sinapizans (H. sect. Sinapizantia) B ITS sequences of H. sect. Naviculospora, rooted with H. islandicum (H. sect. Naviculospora). Hebeloma in Mexico 175 Etymology. From ambustus (Latin adj.) meaning scorched, terra (Latin n.) mean- ing soil and the Latin sufhx -anum indicating position to indicate growing on scorched soil. In Mexico, the local people burn the ground in the pine forests to encourage the growth of this mushroom, which they regard as an excellent edible mushroom. The local people refer to it in Nahuatl as the xolete de ocoxal (or ocoxalnanacatl), the mush- room of the pine needles from Chamusquinero, meaning from burnt ground. Description. Pileus (12) 16-45 (52) mm diameter, usually umbonate or subum- bonate, rarely convex or applanate; margin usually entire, sometimes involute particu- larly when young, often with remains of the universal veil, occasionally spotting, not hygrophanous; usually almost unicolored with color at center usually cream to ochra- ceous or clay-buff but may occasionally be darker, honey to sepia or umber, usually a little paler at the margin. Lamellae emarginate, white, cream to brown, with a weak white fimbriate edge sometimes visible and without droplets, number of full-length lamellae 50-74. Stipe (23) 24-60 (75) mm long, 3-8 (10) mm diameter at median, cylindrical, surface cream, ivory to pale brown but occasionally discoloring from the base upwards, sometimes strongly, fibrillose, at apex pruinose; base with white my- celium. Partial veil present on young specimens, whitish at first, before basidiospores mature, and often clear fibrils remaining on the stipe and pileus. Context in pileus white to cream, firm, in stipe stuffed, becoming hollow with age; taste not recorded, smell occasionally odorless but usually raphanoid, sometimes strongly so or with cacao components. Spore deposit color clay-buff. Basidiospores based on n = 146 spores of the holotype, 5% to 95% percentile range 7.7—9.8 x 5.5—7.0 um, with median 8.9 x 5.9 um and av. 8.9 x 6.0 um with S.D. length 0.68 ym and width 0.44 um; Q value 5% to 95% percentile range 1.25—1.63, with median 1.48 and av. 1.47 with S.D. 0.11; spore size based on 33 collections medians 7.8—10.3 x 5.5—6.4 um and av. 8.0—10.2 x 5.6-6.5 um with av. S.D. length 0.61 um and width 0.35 um, av. Q 1.36 —-1.61, ellipsoid or ovoid, with small apiculus, apex round or subacute, with a distinct thinning of the apical wall, guttulate with one or sometimes more oily drops, usually almost smooth even under immersion, with perispore not loosening, almost totally non-dextrinoid with just an indistinct brown- ish tint in Melzer’s reagent (O1; PO; D1); pale yellow to brown in KOH. Basidia 25— 34 x 6-8 um, with av. Q 3.7-4.4, cylindrical to clavate, hyaline, 4-spored. Cheilocyst- idium width near apex holotype 5% to 95% percentile range 3.5—5.3 um, with median 4.3 um and av. 4.3 um with S.D. 0.63 um; across 33 collections median 4.1—5.4 um and av. 4.1-5.1 um; examining approx. 20 selected cheilocystidia of each of the 33 collections yields a range for the avs. of 35-55 x 4.1—5.1 x 4.2-5.1 x 7.1-9.9 um and 35 x 4.3 x 4.2 x 7.3 um av. for holotype; av. ratios A/M: 0.96—1.15, A/B: 0.51—0.70, B/M: 1.55—2.31, mainly swollen in the lower half, some ventricose or lageniform, often with one or two septa, rarely geniculate or with some thickening of the median wall, hyaline. Pleurocystidia absent. Caulocystidia similar to cheilocystidia but more cylindrical and larger, up to 140 um. Pileipellis an ixocutis; epicutis up to 100 um thick, with gelatinized, often encrusted hyphae up to 6 um wide; subcutis yellow and 176 Ursula Eberhardt et al. / MycoKeys 90: 163-202 (2022) MES es Sew: 1p ees Figure 6. Hebeloma ambustiterranum A-C basidiomata A holotype TLXM 6155 (HJB16802) B TLXM HJB16803. C TLXM HJB16805 D mushroom vendor in the market of Tlaxcala City E H. ambustiter- ranum sold in the market of Tlaxcala City. Photos A-D H.J. Beker E A. Montoya. the trama below the cutis made up of cylindrical or occasionally ellipsoid cells up to 14 um wide. Clamp connections present throughout the basidiome. Ecology and distribution. In temperate coniferous woodlands on burnt ground with Pinus and Quercus. Growth habit usually scattered, rarely solitary or caespitose. To date, all collections of Hebeloma ambustiterranum recorded from Mexico at lati- tudes between 19°N and 20°N and altitudes above 2000 m. Additional collections examined. Mexico. Mexico City: Municipality of Milpa Alta, approx. 19.1942°N, 99.0267°W, alt. approx. 2400 m, 4 Jul 2011, R. Vanegas-En- riquez (TLXM RVE042, HJB17734). Municipality of Milpa Alta, approx. 19.1942°N, 99.0267°W, alt. approx. 2400 m, 16 Jul 2011, R. Vanegas-Enriquez (TLXM RVE049, HJB17735). Municipality of Milpa Alta, approx. 19.1942°N, 99.0267°W, alt. approx. 2400 m, 21 May 2013, A.C. Lopez (TLXM ACL-MA-085, HJB17736). Puebla: Mu- nicipality of Acajete, La Malinche National Park, north of Santa Isabel Tepetzala, ap- prox. 19.1471°N, 97.924°W, alt. approx. 2600 m, on soil in woodland under Pinus sp., 15 Jul 1998, R. Reyes-Lopez (TLXM RL1-01, HJB16780). Municipality of Acajete, La Malinche National Park, 4 km north of Santa Isabel Tepetzala, approx. 19.1471°N, 97.9239°W, alt. approx. 2600 m, on soil in woodland under Pinus sp., 29 Jul 1998, R. Reyes-Lépez (TLXM RL2-7, HJB16765). Tlaxcala: La Malinche National Park, 19.2742°N, 97.9833°W, alt. approx. 2850 m), on burnt soil in coniferous woodland under Pinus montezumae and Pinus teocote, 8 Jul 2017, Forayer (TLXM HJB16799). Hebeloma in Mexico P Figure 7. Holotype of Hebeloma ambustiterranum TLXM 6155 (HJB16802) A basidiospores x1600 B spore ornamentation x1600 C basidiospores in Melzer’s reagent x1600 D=E cheilocystidia x1000 F basidia x1000 G cheilocystidia on lamella edge x500 H caulocystidia x500 I Cutis x125. All in KOH, except C. Scale bars: 5 um (A=F); 10 um (G-J); 50 um (K). Photos H.J. Beker. 178 Ursula Eberhardt et al. / MycoKeys 90: 163-202 (2022) La Malinche National Park, 19.2744°N, 97.9831°W, alt. approx. 2850 m, on burnt soil in coniferous woodland under Pinus montezumae and Pinus teocote, 8 Jul 2017, L. Davies (TLXM HJB16800). La Malinche National Park, 19.2743°N, 97.9829°W, alt. approx. 2840 m 8 Jul 2017, L. Davies (TLXM HJB16801), on burnt soil in co- niferous woodland under Pinus montezumae and Pinus teocote. La Malinche National Park, 19.2749°N, 97.9820°W, alt. approx. 2830 m, 8 Jul. 2017, A. Montoya-Esquiv- el, A. Kong (TLXM HJB16803), on burnt soil in coniferous woodland under Pinus montezumae and Pinus teocote. La Malinche National Park, 19.2752°N, 97.9820°W, alt. approx. 2830 m, on burnt soil in coniferous woodland under Pinus montezumae and Pinus teocote, 8 Jul 2017, A. Kong (TLXM HJB16804). La Malinche National Park, 19.2753°N, 97.9823°W, alt. approx. 2830 m, on burnt soil in coniferous wood- land under Pinus montezumae and Pinus teocote, 8 Jul. 2017, A. Montoya-Esquivel, A. Kong (TLXM HJB16805). La Malinche National Park, 19.2751°N, 97.9825°W, alt. approx. 2830 m, on burnt soil in coniferous woodland under Pinus montezumae and Pinus teocote, 8 Jul 2017, A. Montoya-Esquivel, A. Kong (TLXM HJB16806). La Malinche National Park, 19.2754°N, 97.9824°W, alt. approx. 2830 m, on burnt soil in coniferous woodland under Pinus montezumae and Pinus teocote, 8 Jul 2017, H.J. Beker (TLXM HJB16807). La Malinche National Park, 19.2755°N, 97.983°W, alt. approx. 2830 m, on burnt soil in coniferous woodland under Pinus montezumae and Pinus teocote, 8 Jul 2017, A. Montoya-Esquivel, A. Kong (TLXM HJB16808). La Malinche National Park, 19.2652°N, 97.9744°W, alt. approx. 2825 m, on soil in coniferous woodland ditch under Pinus teocote, 9 Jul 2017, A. Montoya-Esquivel (TLXM HJB16818). Municipality of Huamantla, La Malinche National Park, Los Pilares, approx. 19.3184°N, 97.9233°W, alt. approx. 2500 m, on soil in woodland under Pinus sp., 2 Aug 1991, A. Montoya-Esquivel (TLXM AME1048, HJB16788). Municipality of Nanacamilpa, 19.4925°N, 98.5778°W, alt. approx. 2725 m, on burnt soil and litter in coniferous woodland under Pinus montezumae, 6 Jul 2017, Forayer (TLXM HJB16747). Municipality of Nanacamilpa, 19.4923°N, 98.5783°W, alt. ap- prox. 2730 m, on burnt soil and litter in coniferous woodland under Pinus montezu- mae, 6 Jul 2017, A. Kong (TLXM HJB16748). Municipality of Nanacamilpa, road from Nanacamilpa to Tepuente, 19.4922°N, 98.5783°W, alt. approx. 2730 m, on burnt soil and litter in coniferous woodland under Pinus montezumae, 6 Jul 2017, A. Montoya-Esquivel (TLXM HJB16749). Municipality of Nanacamilpa, road from Nanacamilpa to Tepuente, 19.4928°N, 98.5792°W, alt. approx. 2725 m, on burnt soil and litter in coniferous woodland under Pinus montezumae, 6 Jul 2017, L. Davies (TLXM HJB16750). Municipality of Nanacamilpa, 19.4928°N, 98.5792°W, alt. ap- prox. 2725 m, on burnt soil and litter in coniferous woodland under Pinus monte- zumae, 6 Jul 2017, L. Davies (TLXM HJB16751). Municipality of Nanacamilpa, 19.4933°N, 98.5791°W, alt. approx. 2725 m, on burnt soil and litter in coniferous woodland under Pinus montezumae, 6 Jul 2017, L. Davies (TLXM HJB16752). Mu- nicipality of Nanacamilpa, 19.4935°N, 98.579°W, alt. approx. 2725 m, on burnt soil and litter in coniferous woodland under Pinus montezumae, 6 Jul 2017, L. Davies (TLXM HJB16753). Municipality of Panotla, San Mateo, Huexoyucan, 19.3874°N, Hebeloma in Mexico 179 98.3028°W, alt. approx. 2485 m, on soil in deciduous woodland under Quercus sp., 10 Jul 2017, H.J. Beker (TLXM HJB16820). Municipality of Santa Ana Chiahutem- pan, La Malinche National Park, Surroundings of San Pedro Tlalcuapan, approx. 19.2152°N, 97.9841°W, alt. approx. 3100 m, on soil, 18 Jul 1998, A. Montoya-Es- quivel (TLXM AME1652, HJB16766). Municipality of Tlaxco, north of El Rosario, El Rodeo, approx. 19.3395°N, 99.3605°W, alt. approx. 3100 m, on soil in woodland un- der Pinus sp. and Quercus sp., Jun 1991, A. Kong (TLXM AK1925, HJB16787). Mu- nicipality of Tlaxco, north of El Rosario, El Rodeo, approx. 19.2153°N, 97.9841°W, alt. approx. 3100 m, on soil in woodland under Pinus sp. and Quercus sp., 10 Jul 1991, A. Kong (TLXM AK1972, HJB16790). Municipality of Trinidad Sanchez Santos, La Malinche National Park, east of Javier Mina, approx. 19.2152°N, 97.9841°W, alt. ap- prox. 3100 m, on soil, 21 May 1994, Hernandez-Valencia (TLXM HV6, HJB16778). Municipality of Trinidad Sanchez Santos, La Malinche National Park, east of Javier Mina, approx. 19.2153°N, 97.9841°W, alt. approx. 3100 m, on soil in woodland under Alnus sp. and Pinus sp., 3 Jul 1998, A. Montoya-Esquivel (TLXM AME1643, HJB16781). Tlaxcala City, mushrooms bought at the Tlaxcala market, 10 Jul 1999, A. Montoya-Esquivel (TLXM AME1713, HJB16764). Tlaxcala City, bought in mar- ket at Tlaxcala, collected from La Malinche National Park, 19.3218°N, 98.2387°W, alt. approx. 2160 m, 8 Jul 2017, M.EM. Aguilar (TLXM HJB16809). Tlaxcala City, bought in market at Tlaxcala, collected from La Malinche National Park, 19.3218°N, 98.2388°W, alt. approx. 2160 m, 8 Jul 2017, M.EM. Aguilar (TLXM HJB16810). Remarks. With its small ellipsoid, non-dextrinoid basidiospores and cheilocystidia swollen in the lower half, often lageniform or ventricose, this taxon clearly belongs to Hebeloma sect. Hebeloma and is closely related to the complex of species around H. mesophaeum. The close, but not crowded, lamellae with more than 50 full length lamellae rules out H. excedens and H. mesophaeum, both of which are widespread throughout North America (Eberhardt et al. 2022a). Indeed, were this mushroom collected in Europe, and the key of Beker et al. (2016) applied, this would key out to H. subtortum. Hebeloma subtortum is most common in southern Europe, growing with lowland pines, and not known from North America. Within North America, no known taxon in H. sect. Hebeloma with such small ellipsoid spores has this number of full-length lamellae, making these characters sufficient for its determination. Fig. 6D—E show this mushroom for sale in local markets of Tlaxcala, where it is regarded as a prized edible mushroom known as hongo de ocote (ocote mushroom) in Spanish (Montoya et al. 2002). It is gathered from the temperate pine woodlands at altitudes of 2000 m and above. The local people burn the ground in the pine forests, ahead of the growing season, to encourage the growth of this mushroom. Frequent, controlled fires prevent the development of hot fires that would also dam- age the pines and pine roots, which are required for the fungi to grow. It is referred to in Nahuatl by several names, for example as the Xolete de oc6-xal or oc6-xal- nanacatl (oc6-xalli = pine-litter; mushroom growing in oc6-xal - the mushroom of the pine needles), rastrojo-nanacatl (mushroom growing on stubble), ocochalero, ocotero, ocoxal, ocochal, cholete de ocote, nixtamalero or as chamusquinero, mean- 180 Ursula Eberhardt et al. / MycoKeys 90: 163-202 (2022) ing from burnt ground (Estrada-Martinez et al. 2009; Reyes-Lépez et al. 2020; Viveros-Assad et al. 2019). It is likely the same species as mentioned by Guzman (1977) as “joletes” in Spanish, described as commonly sold in the Amecameca mar- ket, where it is recommended to boil them and then discard the water so that they are safe for consumption. Hebeloma cohaerens A. Montoya & Beker, sp. nov. MycoBank No: 842828 Figs 8-9 Type. Mexico. Tlaxcala: Municipality of Panotla, 1 km al este de San Francisco Tem- ezontla, approx. 19.3496°N, 98.2784°W, alt. approx. 2600 m, in deciduous woodland under Quercus sp., 23 Jul 2017, A. Montoya-Esquivel AME3102 (holotype TLXM 6156; isotype BR 5020224875654V; HJB17733); Genbank ITS ON202511. Diagnosis. The short clavate-ventricose cheilocystidia, with average apical width less 6.5 um, the small (on average less than 10 x 5.5 um), weakly ornamented but rather strongly dextrinoid basidiospores and the whitish to cream or buff color of the pileus, differentiate this species from other Hebeloma species. Etymology. From cohaerens (adj. Latin) meaning united or joined together, to emphasize the connate habitus. Description. Pileus (22) 32-38 (47) mm diameter, convex, often applanate, oc- casionally umbonate; margin smooth, often involute, particularly when young, occa- sionally eroded, not hygrophanous; usually almost unicolored, usually cream or buff, sometimes slightly paler towards margin. Lamellae often adnate or adnexed, occasion- ally emarginate, depth up to 4 mm, white, cream to brown, with white fimbriate edge but without droplets on the lamella edge, number of full-length lamellae 70-80. Stipe (31) 37-46 (48) mm long, (5) 7-8 (10) mm diameter at median, usually cylindrical but sometimes with a clavate base, surface cream, ivory, not discoloring, fibrillose, pru- inose, particularly towards apex; base with white mycelium. Context in pileus and stipe white to cream, firm, in stipe stuffed; taste not recorded, smell earthy. Spore deposit color not recorded. Basidiospores based on n = 64 spores of the holotype, 5% to 95% percentile range 8.6-10.5 x 4.9-5.7 um, with median 9.4 x 5.3 wm and av. 9.5 x 5.3 um with S.D. length 0.57 um and width 0.26 um; Q value 5% to 95% percentile range 1.64—1.95, with median 1.79 and av. 1.78 with S.D. 0.10; spore size based on four collections medians 9.1—9.5 x 5.3-5.6 um and av. 9.1-9.5 x 5.3-5.5 um with av. S.D. length 0.50 um and width 0.30 um, av. Q 1.65-1.78, amygdaloid, occasionally limoniform, with small apiculus and rounded apically, often subacute, with a distinct thinning of the apical wall and sometimes a papilla, usually guttulate with one or sometimes more oily drops, at most weakly ornamented (ornamentation only visible under immer- sion), with a perispore hardly loosening, rather strongly dextrinoid, becoming medium reddish brown in Melzer’s reagent (O1/2; PO; D3); yellow brown in KOH. Basidia Hebeloma in Mexico 181 4 YZ EB . 4 MUTA A i py ar Figure 8. A-B basidiomata, holotype of Hebeloma cohaerens TLXM 6156 (HJB17733). Photos A. Kong. 22-27 x 5-7 um, with av. Q 3.7—3.8 um, cylindrical to clavate, hyaline, 4-spored. Cheilocystidium width near apex holotype 5% to 95% percentile range 4.7—7.7 um, with median 6.0 um and av. 6.1 um with S.D. 1.0 um; across four collections median 5.6—-6.4 um and av. 5.5—6.3 um; examining approx. 20 selected cheilocystidia of each of the four collections yields a range for the avs. of 33-36 x 5.5-6.3 x 3.5-4.1 x 5.5— 6.6 um and 33 x 6.1 x 4.1 x 6.5 um av. for holotype. Cheilocystidium av. ratios A/M: 1.49-1.63, A/B: 0.86-1.03, B/M: 1.59-1.88, mainly clavate-ventricose, often with one or two septa. Pleurocystidia absent. Caulocystidia similar to cheilocystidia but larger, up to 90 um long. Pileipellis an ixocutis with an epicutis up to 110 um thick, with gelatinized, hyphae up to 6 um wide; subcutis cream to pale yellow, and the trama below the cutis made up of cylindrical, often ellipsoid cells, up to 14 um wide. Clamp connections present throughout the basidiome. Ecology and distribution. In deciduous or mixed woodlands apparently associ- ated with Quercus or Pseudotsuga. Growth habit mainly caespitose, sometimes with a few scattered basidiomes. To date, all collections of Hebeloma cohaerens recorded from Tlaxcala at altitudes of 2600 m or more. Additional collections examined. Mexico. Tlaxcala: Municipality of Panotla, 1 km al este de San Francisco Temezontla, approx. 19.3496°N, 98.2784°W, alt. approx. 2600 m, in deciduous woodland under Quercus sp., 23 Jul 2017, A. Montoya-Esquivel (TLXM AME3101, HJB17732). Municipality of Panotla, 1 km al este de San Fran- cisco Temezontla, approx. 19.3496°N, 98.2784°W, alt. approx. 2600 m, in deciduous woodland under Quercus sp., 23 Jul 2017, A. Kong (TLXM AK17-08, HJB17737). Municipality of Terrenate, Rancho el pozo, approx. 19.5407°N, 97.9046°W, alt. ap- prox. 2900 m, on soil in woodland under Pseudotsuga sp., 13 Jul 1995, Galindo-Flores (TLXM GF1866, HJB16779). Remarks. The small, short clavate-ventricose cheilocystidia, together with the small rather smooth, rather strongly dextrinoid basidiospores, support the placement of this species within Hebeloma sect. Theobromina. Within this section the pale cream to buff pileus color together with the caespitose habitus is unique. Ursula Eberhardt et al. / MycoKeys 90: 163-202 (2022) C Figure 9. Holotype of Hebeloma cohaerens TLXM 6156 (HJB17733) A basidiospores x1600- B spore ornamentation x1600 C basidiospores in Melzer’s reagent x1600 D=E cheilocystidia x1000 F basidia x1000 G cheilocystidia x500 H caulocystidia x500 I epicutis hyphae x1000 J subcutis x1000 K cutis x125. All in KOH, except C. Scale bars: 5 um (A=F); 10 um (G=J); 50 um (K). Photos H.J. Beker. Hebeloma in Mexico 183 The description was based on just four collections all from the same region of Mexico, and is not known from any other location. More records for this species will help to define better its morphological characters and its biogeographic preferences. The minimum interspecific distance between the ITS sequences of H. cohaerens and sequences from other species is around 1.2%. The BLAST result of the type sequence of H. cohaerens against UNITE resulted in a hit of a soil sample sequence, pointing towards UNITE SH1563973.08FU (98.5% level). This SH includes two independently generated sequences from California (UDB0767851, soil sample, Tedersoo et al. 2021; DQ822802, basidiome, Point Reyes National Seashore Reserve, under Pinus muricata, Peay et al. 2007) that differ by around 0.5% from the sequences assigned to H. cohaerens, but match no other species. These results suggest that H. cohaerens may occur in the US (California) and the UNITE SH corresponding to H. cohaerens is likely to be SH1563973.08FU. Hebeloma magnicystidiatum A. Kong & Beker, sp. nov. MycoBank No: 842829 Fig. 10 Type. Mexico. Tlaxcala: Municipality of Totolac, Tepeticpac, 19.3457°N, 98.2226°W, alt. approx. 2400 m, on the ground in woodland under Pinus sp. and Quercus sp., 29 Aug. 1990, A. Estrada-Torres AET3093 (holotype TLXM 6157; isotype BR 5020224873599V; HJB16795); GenBank ITS ON202534. Diagnosis. The amygdaloid, non-dextrinoid, rather strongly ornamented spores with average Q value less than 1.6 and the capitate-stipitate cheilocystidia with average width at the apex greater than 9.5 um distinguish this species from all other known Hebeloma species. Etymology. From magni- (Latin, composite) meaning large and cystidiatus to em- phasize the large capitate-stipitate cheilocystidia. Description. Pileus 19-26 mm diameter, convex, surface dry, finely tomentose, cuticle separable, reddish yellow to brown in the center, and pale orange towards the margin. Lamellae emarginate, white, cream to orange brown as the spores mature, with a white fimbriate edge, and about 60 full-length lamellae. Stipe 10-21 mm long, 4—6 mm diameter at median, cylindrical, surface whitish but discoloring brown from the base upwards, with age or handling, fibrillose, at apex pruinose. Context in pileus white to cream, firm, in stipe stuffed, initially white to cream but becoming brown with age and handling, becoming hollow with age; taste fungal to sweet, smell rapha- noid. Spore deposit not recorded. Basidiospores based on n = 44 spores of the holotype, 5% to 95% percentile range 9.7-11.6 x 6.4-7.6 um, with median 10.5 x 7.0 um and av. 10.5 x 7.0 um with S.D. length 0.62 um and width 0.42 um; Q value 5% to 95% percentile range 1.40—1.62, with median 1.49 and av. 1.50 with S.D. 0.07; amygdaloid, often limoniform, with small apiculus and rounded apically, often subacute to acute, with a distinct thinning of the apical wall and sometimes a clearly visible papilla, not guttulate, usually rather 184 Ursula Eberhardt et al. / MycoKeys 90: 163-202 (2022) Figure 10. Holotype of Hebeloma magnicystidiatum TLXM 6157 (HJB16795) A Basidiospores x 1600 B spore ornamentation x1600 C basidiospores and D spore ornamentation in Melzer’s reagent x1600 E basidium x1000 F—H cheilocystidia x1000 H cheilocystidia x500 I caulocystidia x500 J cutis x125 K epicutis hyphae x500 L subcutis x500. All in KOH, except C-D. Scale bars: 5 um (A=G); 10 um (H-I, K-L); 50 um (J). Photos H.J. Beker. Hebeloma in Mexico 185 strongly ornamented, ornamentation visible even without immersion, with perispore at most somewhat loosening in a few spores, an indistinct brownish tint in Melzer’s reagent (O3; P1; D1); yellow-brown in KOH. Basidia 27.5—-35 x 7.5—9 um, with av. Q 3.9, cylindrical to clavate, without pigmentation, 4-spored. Cheilocystidium width near apex holotype 5% to 95% percentile range 6.1—-14.3 um, with median 9.1 um and av. 9.7 um with S.D. 2.61 um; examining approx. 20 selected cheilocystidia of the holotype yields a range for the avs. of 55 x 9.7 x 7.3 x 4.3 um av. and cheilocystidium av. ratios A/M: 2.58, A/B: 2.67, B/M: 0.95; mainly capitate-stipitate, unfortunately many collapsed in exsiccata. Pleurocystidia absent. Caulocystidia similar to cheilocys- tidia but larger, up to 80 um long. Pileipellis an ixocutis; epicutis up to 110 um thick, with gelatinized hyphae up to 7 um wide; subcutis yellow; and the trama below the cutis made up of cylindrical or occasionally ellipsoid cells up to 17 um wide. Clamp connections present throughout the basidiome. Ecology and distribution. In woodland on the ground with Comarostaphylis and Quercus. Growth habit scattered. To date, with only one collection of this species, not possible to describe its distribution and ecology. Remarks. With its amygdaloid, hardly dextrinoid basidiospores and capitate- stipitate cheilocystidia, morphologically this taxon clearly belongs to Hebeloma sect. Denudata and there to H. subsect. Crustuliniformia. The amygdaloid spores with rather small average Q value separate this species from all other studied Hebeloma from our database with more than 10,000 collections. While this may suggest that this is a rare species, we have insufficient Hebeloma collections from Mexico to reach such a con- clusion. The single collection was collected in the 1990s, thus the only loci we could amplify were ITS and mitSSU variable regions V6 and V9. The phylogenetic placement of H. magnicystidiatum within H. sect. Denudata is unresolved. As pointed out before (e.g. Eberhardt et al. 2016, 2022b; Beker et al. 2016), the more species rich subsections of H. sect. Denudata (H. subsects Clep- sydroida and Crustuliniformia) are not supported molecularly. In terms of ITS, the most similar species was H. sordidulum (H. subsect. Clepsydroida) with similarity values < 98.7%. Possibly H. magnicystidiatum will correspond to a UNITE SH at the 99% or 98.5% level once sequences of this species are included in the system. Morphologically, the capitate-stipitate cheilocystidia together with the amygdaloid spores with av. Q less than 1.6 are sufficient characters to separate this species from members of H. sect. Clepsydroida, such as H. cavipes, H. matritense, H. sordidulum and H. vaccinum. Hebeloma neurophyllum G.F. Atk., Annales Mycologici 7(4): 370 (1909) Figs 11-12 Type. USA. New York: Coy Glen, Ithaca, approx. 42.4272°N, 76.5241°W, alt. approx. 125 m, on soil in woodland, 18 Oct 1906, N. Coil (holotype CUP-A-021514; isotype TENN-F-037531, HJB1000453, isotype WTU-F-039596, HJB1000558). 186 Ursula Eberhardt et al. / MycoKeys 90: 163-202 (2022) Diagnosis. Gregarium 7—8 cm altum, pileo 5—6 cm lato, stipite 5—6 mm crasso: Pileo ochraceo-cremeo vel fulvo-ochraceo, leviter viscido. Lamellis 8 mm latis, pallide cinnamomeo-rufis, late sinuatis, adnexis, costatis. Basidiis 4-sporis. Sporis subfusoid- eis, 12-15 x 7-8 p[m]. Ad terram in silvis, Ithacae, N. Y. Stipite albo, fibroso-striato, cavo vel subfarcto. English translation of diagnosis. Gregarious 7-8 cm high, pileus 5—6 cm broad, stipe 5—6 mm thick: pileus ochraceous-cream or fulvous-ochraceous, slightly viscid. Lamellae 8 mm broad, pale cinnamon-reddish, broadly sinuate, adnexed, intervenose. Basidia four-spored. Spores subfusoid, 12-15 x 7-8 um. On the ground in woodland, New York. Stipe white, fibrous-striate, fistulose or almost stuffed. Description. Pileus (26) 30-55 (60) mm diameter, convex, occasionally umbonate or broadly umbonate; margin often smooth, occasionally involute or wavy, not hygroph- anous; usually unicolor, occasionally two colors, at center occasionally yellowish brown, ochraceous or cream, rarely fawn, cinnamon or clay-buff, sometimes slightly paler to- wards margin. Lamellae usually emarginate, occasionally adnexed, depth up to 9 mm, white, cream to brown, usually with white fimbriate edge, usually without droplets on the lamella edge but rarely some drops may be visible, number of full-length lamellae 70-94. Stipe (25) 31-75 (80) mm long, 5—14 (16) mm diameter at median, often clavate or bulbous, occasionally cylindrical, (7) 9-16 (18) mm wide at base, surface cream, ivory, rarely discoloring, occasionally velutinous, floccose or fibrillose, often pruinose, particu- larly towards apex. Veil not observed. Context in pileus white to cream, firm, in stipe usu- ally hollow, rarely with superior hanging wick; taste mild, smell occasionally raphanoid or odorless, rarely fruity or earthy. Spore deposit yellowish brown to brownish olive. Basidiospores based on n = 70 spores of the holotype, 5% to 95% percentile range 12.7-15.6 x 7.2—9.0 um, with median 14.2 x 8.2 um and av. 14.2 x 8.2 um with S.D. length 0.93 um and width 0.54 um; Q value 5% to 95% percentile range 1.52-1.91, with median 1.74 and av. 1.73 with S.D. 0.12; spore size based on 47 col- lections medians 11.6—14.3 x 7.2—8.2 um and av. 11.7—14.2 x 7.5-8.3 pm with av. S.D. length 0.898 um and width 0.459 um, av. Q 1.53-1.78, amygdaloid, usually limoniform, with small apiculus and rounded apically, often subacute to acute, with a distinct thinning of the apical wall and a clear papilla, occasionally guttulate with one or sometimes more oily drops, distinctly to strongly ornamented (ornamentation vis- ible without immersion), with a perispore somewhat to distinctly loosening, at least in a few spores, strongly dextrinoid, becoming at least medium brown and often intensely red-brown in Melzer’s reagent (03/4; P1/2; D3/4); yellow to brown in KOH. Basidia 20-43 x 7-10 um, with av. Q 2.7—3.8 um, cylindrical to clavate, with a median con- striction, hyaline, 4-spored. Cheilocystidium width near apex holotype 5% to 95% percentile range 4.9—9.0 um, with median 6.5 um and av. 6.7 um with S.D. 1.27 um; across 47 collections median 4.5—6.8 pm and av. 4.6—6.7 um; examining approx. 20 selected cheilocystidia of each of the 47 collections yields a range for the avs of 40— 59 x 4.6-6.7 x 4.4—5.7 x 5.6-8.4 um and 49 x 6.7 x 5.6 x 6.7 um av. for the holotype. Cheilocystidium av. ratios A/M: 1.01-1.41, A/B: 0.68-1.23, B/M: 1.16-1.58, mainly gently clavate or ventricose, occasionally cylindrical, lageniform or clavate-lageniform Hebeloma in Mexico 187 — ~ , — Figure | 1. Hebeloma neurophyllum, basidiomata A HJB16991 B HJB18101. Photos H.J. Beker. or clavate-ventricose, often with one or two septa, sometimes clamped, often with plaques on the cystidial walls, occasionally geniculate or with basal wall thickening, rarely bifurcate, hyaline, rarely with yellow contents. Pleurocystidia absent. Caulocyst- idia similar to cheilocystidia but larger, up to 115 um long. Pileipellis an ixocutis, epi- cutis up to 90 um thick, with gelatinized, hyphae up to 6 um wide; subcutis pale yellow to brownish yellow, and the trama below the cutis made up of cylindrical, often ellip- soid cells, up to 16 um wide. Clamp connections present throughout the basidiome. Habitat and distribution. Based on almost 50 collections, where only one pos- sible associate was recorded, the most commonly recorded associates were Picea and Quercus, but Populus, Salix and. Tilia were also recorded; the most commonly recorded families were Fagaceae, Pinaceae and Salicaceae, but Betulaceae and Malvaceae were also recorded. We have additional records where Alnus, Arctostaphylos, Betula, Dryas, Pinus and Polygonum were recorded as possible associates, but in each of these cases a number of possible associates were mentioned. All records of H. neurophyllum are from Northern America, where it is widespread across the region but primarily collected in temperate to boreal woodland, occasionally in urban areas. Additional material examined. Canapa. Alberta: Moose Hill, Breton, Edmon- ton, 53.1418°N, 114.6097°W, alt. approx. 810 m, on soil in mixed woodland under Picea mariana, 12. Aug 2017, H.J. Beker (HJB16856). Northwest Territories: Highway 3, between Yellowknife and Behchoko, 62.5198°N, 114.897°W, alt. approx. 165 m, on mossy soil in boreal, calcareous woodland roadside under Betula sp. and Salix sp., 7 Sep 2018, H.J. Beker, L. Davies (HJB18101). Yukon: Railway Station, White- horse, 60.7214°N, 135.0505°W, alt. approx. 665 m, on soil and litter in boreal shrub- land riverside under Populus tremuloides and Salix sp., 31 Aug 2018, H.J. Beker, L. Davies (HJB17975). 3" Avenue near Wood St intersection, Whitehorse, 60.7212°N, 135.0555°W, alt. approx. 665 m, on grassy, mossy soil in boreal urban roadside under Populus sp., 1 Sep 2018, H.J. Beker, L. Davies (HJB17981). MEXICO. Chihuahua: El Ranchito, approx. 28.3387°N, 105.4076°W, alt. approx. 1150 m, on soil in montane, subtropical woodland, 18 Aug 2001, A. Kong 3782 (TLXM AK3782, HJB16773). UNITED STATES. Alaska: Kantishna Roadhouse Nature Trail, Denali National Park, 188 Ursula Eberhardt et al. / MycoKeys 90: 163-202 (2022) F-037531 (HJB1000453) x1600 C basidiospores of HJB17897 in Melzer’s reagent x1600 D basidium of isotype x1000 E=F cheilocystidia of isotype x1000 G caulocystidia of HJB17975 x500 H caulocystidia of HJB16856 x500 I epicutis hyphae and J subcutis of isotype x500. All in KOH, except C. Scale bars: 5 um (A=+F); 10 um (G-J). Photos H.J. Beker. Hebeloma in Mexico 189 63.5243°N, 150.9625°W, alt. approx. 490 m, on sandy soil in boreal, mixed but mainly coniferous woodland pathside under Alnus sp., Betula sp. and Salix sp., 18 Aug 2018, H.]J. Beker, L. Davies (DENA-61424, HJB17897). Texas: Jefferson County, Beaumont, residence of Penny Clark, approx. 30.0788°N, 94.1372°W, alt. approx. 0 m, in garden under Quercus fusiformis, 4 Dec 2015, D. Lewis DPL11907 (HJB15699). Wiscon- sin: Bark Point Road, near Bark Bay, 46.8353°N, 91.2594°W, alt. approx. 185 m, on grassy soil in coniferous garden under Picea glauca, 13 Sep 2017, L. Davies, H.J. Beker (HJB16991). Remarks. With the mixture of gently clavate and ventricose cystidia alongside the strongly dextrinoid basidiospores, this species belongs within Hebeloma sect. Velutipes. Within this section the combination of spores with minimum average width 7.5 um and a distinctly loosening perispore in at least some spores, together with the absence of pleurocystidia, defines this species. The collection of H. neurophyllum from Mexico, gathered at El Ranchito in Chihuahua, matches well with other collections of this spe- cies. We are not aware of any synonyms for this species. In terms of ITS, the most similar to H. neurophyllum were H. celatum, H. erebium and H. quercetorum, the ITS sequences of which were around 99% similar (99.2— 98.6%) to those of H. neurophyllum. Hebeloma neurophyllum appears to correspond to UNITE SH1733487.08FU (99%). Intriguingly, this species hypothesis includes a number of soil sample sequences from Estonia, suggesting that either H. neurophyllum occurs in Europe, too, or that species known to occur in Europe also contain ITS cop- ies corresponding to H. neurophyllum below the detection limit of Sanger sequencing. Hebeloma subaustrale Murrill, Lloydia 8: 287 (1946) [1945] Fig. 13 = Hebeloma angustisporium Hesler, Kew Bulletin 32(3): 471 (1977) = Hebeloma perangustisporium Hesler, Kew Bulletin 32(3): 478 (1977) Type. USA. Florida: Gainesville, Alachua Co., approx. 29.651634°N, 82.324826°W, alt. approx. 50 m, on grassy, shady soil in lawn, 30 Oct 1941, G.E Weber (holotype FLAS-F-19345, HJB1000402; isotype TENN-F-021177, HJB1000447). Diagnosis. Pileo convexo-expanso, 3—4 cm. lato, subviscido, glabro, pallido-ro- seo, raphanico; lamellis sinuatis, latis, confertis; sporis subovoidcis, pallidis, levibus, 8-10 x 4—4.5 p[m]; stipite acquali, pallido, 3 x 0.5 cm. English translation of diagnosis. Pileus convex to applanate, 3-4 cm broad, slight- ly viscid, glabrous, pale pink, with raphanoid smell; lamellae sinuate, broad, crowded; spores subovoid, pale, smooth, 8-10 x 4—4.5 u[m]; stipe equal, pale, 3 x 0.5 cm. Description. Pileus (20) 32-45 (46) mm diameter, usually convex, occasionally umbonate; occasionally with remains of universal veil; margin often smooth, occasion- ally scalloped, not hygrophanous; usually unicolor, occasionally two colors, at center cream to buff to ochraceous, often becoming paler towards the margin. Lamellae usu- 190 Ursula Eberhardt et al. / MycoKeys 90: 163-202 (2022) ally emarginate, occasionally adnate or adnexed; white, cream to brown, usually with white fimbriate edge, without droplets on the lamella edge, number of full-length lamellae 80—92. Stipe 30-56 (70) mm long, 5—10 (11) mm diameter at median, often clavate or cylindrical, 5-13 (14) mm wide at base, surface cream, ivory to white rarely discoloring, pruinose, particularly towards apex. Context in pileus white to cream, firm, similar color in stipe, becoming hollow with age; taste raphanoid, smell rapha- noid, occasionally earthy. Spore deposit cinnamon color. Basidiospores based on n = 63 spores of the holotype, 5% to 95% percentile range 8.4—9.8 x 4.6—-5.2 um, with median 9.0 x 4.8 um and av. 9.0 x 4.9 um with S.D. length 0.51 um and width 0.18 um; Q value 5% to 95% percentile range 1.65—2.03, with median 1.88 and av. 1.85 with S.D. 0.12; spore size based on seven collections medians 8.5-10.2 x 4.6-5.3 um and av. 8.6-9.9 x 4.6—5.3 um with av. S.D. length 0.657 um and width 0.271 um, av. Q 1.73—2.09, amygdaloid, usually fusoid, rarely navicular, with small apiculus and rounded apically, often subacute to acute, with a distinct thin- ning of the apical wall and no papilla, occasionally guttulate with one or sometimes more oily drops, very weakly ornamented (ornamentation only visible under immer- sion), with a perispore somewhat loosening, in at most a few spores, rarely not loosening or distinctly loosening, distinctly to rather strongly dextrinoid, becoming yellow brown to medium brown in Melzer’s reagent (O1/2; P0/1/2; D2/3); yellow in KOH. Basidia 19-32 x 5-7 um, with av. Q 3.8-4.6 um, cylindrical to clavate, hyaline, 4-spored. Cheilocystidium width near apex holotype 5% to 95% percentile range 4.5—6.8 um, with median 5.8 um and av. 5.7 wm with S.D. 0.85 um; across seven collections median 4,.4—6.3 um and av. 4.5-6.3 um; examining approx. 20 selected cheilocystidia of each of the seven collections yields a range for the avs of 29-43 x 4.5-6.3 x 3.9-5.1 x 4.8- 6.8 um and 33 x 5.7 x 4.3 x 5.6 um av. for the holotype. Cheilocystidium av. ratios A/M: 1.04—-1.48, A/B: 0.84-1.31, B/M: 1.20—1.36, irregular but mainly cylindrical, often ventricose, often clavate, occasionally clavate-lageniform or clavate-ventricose or gently clavate, rarely capitate stipitate or clavate stipitate, often with one or two septa, occasionally with apical wall thickening. Pleurocystidia absent. Caulocystidia similar to cheilocystidia but larger, up to 100 um. Pileipellis an ixocutis, epicutis up to 100 um thick, with gelatinized, hyphae up to 7 um wide, often encrusted; subcutis pale yellow; and the trama below the cutis made up of ellipsoid or thickly sausage-shaped, often cy- lindrical cells up to 13 um wide. Clamp connections present throughout the basidiome. Habitat and distribution. Where only one possible associate was recorded, that associate has always been Quercus (Fagaceae). We have additional records where Pi- nus, Abies and Fagus were recorded as possible associates, but in each of these cases a number of possible associates were mentioned by the collector. We are only aware of five collections other than that from Mexico. These are all from the eastern half of the United States: Ohio, Pennsylvania and Tennessee. Additional material examined. Mexico. Tlaxcala: Municipality of Huamantla, La Malinche National Park, Cafada Grande, east side of La Malintzi volcano, ap- prox. 19.1999°N, 97.9729°W, alt. approx. 3000 m, on soil in montane, temperate woodland under Abies sp. and Pinus sp., 25 Jul 1990, H. Cuevas HC1155 (TLXM Hebeloma in Mexico 191 , o i Figure 13. Hebeloma subaustrale A basidiospores and B spore ornamentation of holotype FLAS-F-19345 x1600 C spores of SPFS-2011-63 (HJB17796) in Melzer’s reagent x1600 D spores and cheilocystidia of holotype x1000 E cheilocystidia of isotype TENN-F-021177 x1000 F cheilocystidia of holotype x1000 G caulocystidia of isotype x1000 H cutis of SPFS-2011-63 x125. All in KOH, except C. I basidiomata of collection SPFS-2011-63. Scale bars: 5 um (A=G); 50 um (H). Photos A=G H.J. Beker H D. Bartholow. 192 Ursula Eberhardt et al. / MycoKeys 90: 163-202 (2022) Table |. Comparison of the most taxonomically important holotype characters of Hebeloma subaustrale and its synonyms. Macroscopic data from the original descriptions and microscopic measures from own studies. Species Hebeloma angustisporium — Hebeloma perangustisporium Hebeloma subaustrale Number of complete lamellae 86 80 88 Spore ornamentation Ol; O2 O2 Ol Spore perispore loosening Pl P1; P2 PO; Pl Spore dextrinoidity D2; D3 D1; D2 D2 Spore length av. (um) 8.6 9.9 9 Spore width av. (um) 5, 5.3 4.9 Spore Q av. 1.73 1.87 1.85 Cheilocystidia length av. (um) 29 39 33 Cheilocystidia apex on gill edge av. (um) 4.5 4.6 5.7 Cheilocystidia av. Q1, A/M 1.04 1.12 1.38 Cheilocystidia av. Q2, A/B 0.86 0.84 1.06 Cheilocystidia av. Q3, B/M 1.24 1.36 1.44 Basidia Q av. 4.3 3.8 3.8 Pileus diameter (mm) 25-40 20-45 30-40 Stipe median width (mm) 9-10 9-11 5 HC1155, HJB16793). USA. Ohio: Shaker Parklands, Doan Brook Gorge, approx. 41.495°N, 81.5953°W, alt. approx. 275 m, on grassy soil under Fagus sp. and Quercus sp., 26 Sep 2011, D. Bartholow SPFS-2011-63 (HJB17796). Pennsylvania: Fort Washington Park, Parking Lot 5, approx. 40.1208°N, 75.2232°W, alt. approx. 80 m, on soil in mixed woodland under Quercus sp., 23 Oct 2018, T. Deluce (HJB18418). Tennessee: Gatlinburg, Great Smoky Mountains National Park, Indian Gap, approx. 35.6108°N, 83.4386°W, alt. approx. 1650 m, 29 Jul 1941, L.R. Hesler LRH13890 (holotype of Hebeloma perangustisporium TENN-F-013890, HJB1000450). Blount, Townsend, Great Smoky Mountains National Park, Cades Cove, approx. 35.6019°N, 83.8113°W, alt. approx. 550 m, 23 Aug 1959, L.R. Hesler LRH23364 (holotype of Hebeloma angustisporium TENN-F-023364, HJB1000314). Remarks. ‘The small weakly ornamented basidiospores together with the short ir- regular cheilocystidia, often cylindrical but also both ventricose and clavate, suggest Hebeloma sect. Naviculospora, which is supported by molecular data. Within this sec- tion H. subaustrale is differentiated from other Northern American species of this sec- tion by the average basidiospore length (a maximum of 10 um), and average spore Q greater than 1.7, together with the cheilocystidia that have a maximum average A/B ratio of 1.5 and a minimum average B/M ratio of 1.2. We were not able to generate any sequence data from the type of H. subaustrale. However, our morphological study of the type, and of a number of other species within H. sect. Naviculospora, leaves us in no doubt that this is a conspecific of both H. an- gustisporium and H. perangustisporium. For these latter two species types we have good morphological and molecular data. Table 1 shows a comparison of the most important taxonomic parameters for the holotypes of these three species. The spore size and the average cheilocystidium shape, despite their irregularity, are key to differentiating spe- cies within this section. The Mexican collection corresponded well with this type mate- rial and other recent collections from the USA. Hebeloma in Mexico 193 Hebeloma subaustrale formed a reasonably well supported clade in the ITS analysis (Fig. 5B), thus it is expected to be identifiable by its barcode. Although the maximum intraspecific distance of the sequences in the analysis is only 0.14%, the minimum distance to other species of the section is 0.7%. At this time (4 Feb 2022), there is no multi-sequence UNITE SH that represents the species; the published sequence of the holotype of H. angustisporium (NR_119890 = HQ179674) formed a singleton SH at the 99% level and the respective SH at the next level included several species. Discussion The systematic position of the discussed species, three new (H. ambustiterranum, H. cohaerens and H. magnicystidiatum) and two neglected and rediscovered (H. neuro- phyllum, H. subaustrale), are unambiguous and supported by morphological and mo- lecular results. All species can be placed in previously described sections of Hebeloma. Based on our current knowledge, all species are easy to delimit molecularly and are recognizable by their [TS-barcodes. Garibay-Orijel et al. (2013) identified H. albocolossum (synonymized with H., eburneum by Beker et al. 2016), H. helodes, H. leucosarx and H. mesophaeum from ectomycorrhizal root tips of Pinus montezumae from the Transmexican Volcanic Belt, based on the sequences available in GenBank at the time. The sequences of Garibay- Orijel et al. (2013) were not included in the tree analyses, because ITS only entries would have negatively influenced the phylogenetic resolution of the respective analyses. Based on currently available sequence data, we would tend to identify the sequenc- es obtained in that study as H. eburneum (JN704820; species in Fig. 3), H. excedens or H. mesophaeum (JN704814; species in Fig. 2), H. velutipes (JN704825; species in Fig. 5), and H. sordidulum (JN704810; species in Fig. 3). These species are treated in detail by Beker et al. (2016) and Eberhardt et al. (2021a, 2022a). Given that these iden- tifications are based only on ITS sequence data, they have to be treated with caution. Many of the issues such as conflicting phylogenetic hypotheses or lack of species resolution in phylogenetic analyses have been encountered and discussed before for H. sect. Denudata (Eberhardt et al. 2015, 2016a; Beker et al. 2016), for H. sect. Ve- lutipes (Aanen et al. 2001; Grilli et al. 2016; Beker et al. 2016) and H. sect. Hebeloma (Beker et al. 2016; Eberhardt et al. 2022a). For the delimitation and recognition of the species described in detail here, H. ambustiterranum, H. cohaerens, H. magnicystidi- atum, H. neurophyllum and H. subaustrale, these are non-issues. For H. magnicystidi- atum the conflicts between the different loci used imply that there was no molecular support for the assignment to subsection. However, already Eberhardt et al. (2016a) showed that even when using additional loci such as RPB2, TEFla and MCM7 sup- port for H. subsects. Clepsydroida and Crustuliniformia was lacking and their relation to H. subsect. Hiemalia was unresolved. Likewise, Grilli et al. (2016) showed that the phy- logenetic relationship between H. celatum, H. erebium and H. quercetorum could not be resolved based on five loci. Here, H. neurophyllum is presented as a fourth species in this group the evolutionary history of which could not be reconstructed based on four loci. 194 Ursula Eberhardt et al. / MycoKeys 90: 163-202 (2022) Other questions arising from the presented results will have to be tackled in a wider context with more samples, more loci and geographically wider sampling. These include whether H. excedens and H. mesophaeum should be treated as a single species (see also Eberhardt et al. 2022a), or whether to attach any importance to the somewhat isolated position of the Mexican H. eburneum in relation to other H. eburneum sequences in the analysis, or the divergent mitSSU V6 sequences of Mexican H. velutipes. Eberhardt and co-workers (2016) showed that member species of the H. alpinum complex varying in their mitSSU variable regions are likely to belong to different mating groups defined by Aanen and Kuyper (1999). Using the same reasoning, if the mitSSU V6 differences of the Mexican H. eburneum or H. velutipes had been accompanied by morphological dif- ferences, we would have had to recognize them as a distinct species. There were no dif- ferences found, thus the collections are here addressed as H. eburneum and H. velutipes, respectively, although the suspicion remains that the mitSSU results point towards mat- ing groups—and possibly species—so far not sampled outside Mexico. Or, alternatively, that our current concept recognizes too many species in the respective groups. There have been reports of edible Hebeloma species from other regions of the world, for example from Guatemala, Laos and Nigeria (Aremu et al. 2009; Carrasco- Hernandez et al. 2015; Eberhardt et al. 2020a; Flores-Arzui 2020), where, for example, Eberhardt and colleagues reported that in Laos H. parvisporum is sold in markets and on roadsides as edible and that it is called “wai khom,” which refers to its bitter taste, which, apparently, remains, at least to some degree, after cooking. From their literature review, Carrasco-Hernandez et al. (2015) found that cytotoxic triterpenes, lanostanetype triterpene esters, neurotoxic cucurbitane-type glycosides and 6,7-seco-caryophyllenes, and related sesquiterpenoids may be the cause of Hebeloma toxicity. It is reported that Hebeloma poisonings typically cause gastrointestinal symp- toms in humans that pass after several days. It is not known which species of Hebeloma are poisonous, but, as said above, their consumption is strongly discouraged (Bresinsky and Besl 1990, Benjamin 1995). It was pointed out (Beker et al. 2016; Eberhardt et al. 2020a) that, given the difficulty of species identification within the genus, one could not be certain which toxic compounds referred to which species. Carrasco-Hernandez et al. (2015) described Hebeloma spp. obtained from the Ozumba market, thus presumably intended for human consumption. They recognize three different species, identified as H. alpinum, H. leucosarx and H. mesophaeum. These identifications have to be treated with caution. Certainly, the basidiospore meas- ures they give for H. alpinum and H. leucosarx would appear too small for those species as we interpret them today. The fact that the spore sizes they give for all three species differ considerably from the spore size of H. ambustiterranum would suggest that more than one species of Hebeloma is consumed in Mexico. Hebeloma ambustiterranum is a species of great cultural significance in central Mex- ico, since it is used as food for the preparation of several local recipes. It is commonly and widely sold in local food markets. Traditional management practices are carried out to encourage the production of basidiomes, such as the use of fire. Traditional names have been assigned to the edible taxa of the genus, and it appears that their dis- Hebeloma in Mexico 195 tribution is wide. However, the analysis of a far greater number of samples is required before the real diversity of this group of species may be known and the knowledge of the edible mushrooms of Mexico expanded. Hebeloma species have been considered as “early-stage [ectomycorrhizal] fungi” (Deacon et al. 1983; Mason et al. 1983; Gryta et al. 1997) and gained a reputation as nursery fungi (e.g., Castellano and Molina 1989; Menkis and Vasaitis 2011). There are other species in the genus, further to H. ambustiterranum, known to associate with burnt ground (Beker et al. 2016). High pH and nutrient levels are associated both with nurseries and burnt ground. It is not clear whether H. ambustiterranum occurs in nurseries. However, should H. ambustiterranum be considered for nursery typo utiliz- ing edibles, knowing about the fire ecology should be helpful in establishing inoculum production and stabilizing H. ambustiterranum populations in the long-term. While this study was limited with regard to collecting sites and the number of collections studied, nevertheless, with eleven species new to Mexico, it provides an important step in the understanding of the Hebeloma of Mexico and a basis for fur- ther development. Given how little we know about Hebeloma of Mexico, it appears premature to attempt a key. In lieu of a key for Hebeloma in Mexico (which would be deficient, based on too few collections), we refer to an interactive identification tool for Hebeloma that is currently under development (Bartlett et al. 2021, accepted). Acknowledgements We are very much obliged to A. Bogaerts and P. Ballings of the Botanic Garden Meise (BR) for help with handling various loans from a variety of herbaria. We also thank these herbaria for their help: AH, C, DAOM, DBG, DENA, DUKE, G, H, K, KRAM, L, LIP, LOD, LY, MAK, MICH, MONT, NY, PDD, PRM, ROHB, SWGC, TENN, TLXM, TNS, TURA, UPS and WTU. 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Herbarium abbreviations follow Index Herbariorum (http://sweetgum.nybg.org/science/ih/) and are separated from the specimen numbers by a space or by a hyphen. MuOb, Mushroom Observer https://mushroomobserver.org/. HJB, personal collection of H.J. Beker unless pre- ceded by an herbarium abbreviation. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.90.85267.suppl1 Supplementary material 2 Alignments and trees Authors: Ursula Eberhardt, Alejandro Kong, Adriana Montoya, Nicole Schiitz, Peter Bartlett, Henry J. Beker Data type: Txt file. Explanation note: This file includes all alignments and trees, including single locus trees associated with Eberhardt et al. (2022) Not (only) poison pies — Hebeloma (Hymenogastraceae, Agaricales) in Mexico. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.90.85267.suppl2