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Research Article
Exploring diversity within the genus Tulostoma (Basidiomycota, Agaricales) in the Pannonian sandy steppe: four fascinating novel species from Hungary
expand article infoPéter Finy‡§, Mikael Jeppson|, Dániel G. Knapp‡, Viktor Papp#, László Albert§, István Ölvedi§, Károly Bóka‡, Dóra Varga‡, Gábor M. Kovács‡, Bálint Dima‡
‡ Eötvös Loránd University, Budapest, Hungary
§ Hungarian Mycological Society, Budapest, Hungary
| University of Gothenburg, Göteborg, Sweden
¶ Linnaeus University, Växjö, Sweden
# Hungarian University of Agriculture and Life Sciences, Budapest, Hungary

Corresponding author: Bálint Dima ( cortinarius1@gmail.com )

Academic editor: María P. Martín
© 2023 Péter Finy, Mikael Jeppson, Dániel G. Knapp, Viktor Papp, László Albert, István Ölvedi, Károly Bóka, Dóra Varga, Gábor M. Kovács, Bálint Dima.
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.
Citation: Finy P, Jeppson M, Knapp DG, Papp V, Albert L, Ölvedi I, Bóka K, Varga D, Kovács GM, Dima B (2023) Exploring diversity within the genus Tulostoma (Basidiomycota, Agaricales) in the Pannonian sandy steppe: four fascinating novel species from Hungary. MycoKeys 100: 153-170. https://doi.org/10.3897/mycokeys.100.112458
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Abstract

Steppe vegetation on sandy soil in Hungary has recently been revealed as one of the hot spots in Europe for the stalked puffballs (genus Tulostoma). In the framework of the taxonomic revision of gasteroid fungi in Hungary, four Tulostoma species are described here as new to science: T. dunense, T. hungaricum, T. sacchariolens and T. shaihuludii. The study is based on detailed macro- and micromorphological investigations (including light and scanning electron microscopy), as well as a three-locus phylogeny of nrDNA ITS, nrDNA LSU and tef1-α sequences. The ITS and LSU sequences generated from the type specimen of T. cretaceum are provided and this resolved partly the taxonomy of the difficult species complex of T. aff. cretaceum.

Key words

Gasteroid, hot spot, molecular systematics, Pannonian inland sand dune thicket, phylogeny, taxonomy, Tulostomataceae

Introduction

The genus Tulostoma was erected by Persoon (1794, 1801) encompassing two species, T. brumale and T. squamosum. Several new species have since been added from all continents, except Antarctica. In a monograph of the genus based on type studies, Wright (1987) accepted 139 species worldwide. Later studies generally confirmed those species concepts, nevertheless reduced some of the species to synonymy (e.g. Moreno et al. (1992, 1997); Altés et al. (1999); Jeppson et al. (2017)). With the introduction of molecular methods in taxonomy, unexpected species diversity has been detected and new, formerly unknown species have been described. In Europe, Jeppson et al. (2017) suggested Mediterranean grassland regions of the Iberian Peninsula, as well as steppe habitats in East Central Europe, as hot-spot areas for species diversity in Tulostoma. Jeppson et al. (2017) described two novel species with type localities in Central Hungary (T. grandisporum, T. pannonicum), but their phylogenetic and morphological results indicated the presence of at least 19 previously-undescribed European species of Tulostoma, nine of which had been collected in Hungary. The species diversity in Eastern Europe was further emphasised by Rusevska et al. (2019) who reported four species from North Macedonia distinct from all known and described species.

In Europe, Hungary has an exceptionally large diversity of gasteroid taxa mainly due to the suitable habitats of the Pannonian sandy steppe areas of the country (Fig. 1). The Festucetum vaginatae plant communities are characteristic on open, continental sandy soils, dominated by the grass species Festuca vaginata which also occur on open steppe mosaics between the poplar–juniper sand dune thickets (Bölöni et al. 2011; Rimóczi et al. 2011). Gasteroid fungi occur especially in those areas where Stipa borysthenica, Fumana procumbens or Juniperus communis are present (Fig. 1).

Figure 1. 

Habitats of Tulostoma species in Hungary: a T. hungaricum in Orgovány b T. sacchariolens in Orgovány c T. dunense in Izsák (Soltszentimre) d T. shaihuludii in Izsák (Soltszentimre). Photos: P. Finy.

In this paper, we propose four species of Tulostoma new to science, two of which were retrieved previously by Jeppson et al. (2017) as Tulostoma sp. 1 and T. aff. cretaceum. Additionally, we present two further species identified through subsequent collections and field investigations. One of the new species was previously reported from Hungary under the name T. volvulatum (Hollós 1904; Siller and Vasas 1995; Siller et al. 2005) and T. obesum (Siller et al. 2006; Rimóczi et al. 2011) which is listed as a protected species by Hungarian law.

Materials and methods

Samples of Tulostoma were collected in Hungary over a period of more than 25 years. Collecting has mostly been performed in the sandy habitats of the Great Hungarian Plain on both sides of the Danube (Kiskunság, Mezőföld). Studied collections were deposited in the herbaria BP (only holotypes), GB and in the Department of Plant Anatomy, Eötvös Loránd University (abbreviated further as ELTE).

Macromorphological study

Mature basidiomata of Tulostoma were collected and studied under a stereomicroscope, regarding their macromorphological characteristics (size, colour, shape of the spore-sac (capitulum), type of mouth (ostiole), type of exoperidium as well as features of the stem), in accordance with Wright (1987). In situ or ex situ photo-documentation of each sample was carried out.

Microscopy

Microscopic features were studied under an Olympus BH-2 light microscope. Samples were mounted in lactophenol-cotton blue and heated to boiling temperature. Measurements were performed under 1000× magnification and calculated digitally using Piximètre software (www.piximetre.fr). Spore dimensions are given without the ornamentation of the spore walls. Small pieces of peridium and gleba from dried basidiomata were prepared, fixed to stubs, coated with gold and examined under a Hitachi S2460N (Hitachi Ltd., Tokyo, Japan) scanning electron microscope (SEM) at 22 kV accelerating voltage.

Molecular study

Total DNA extraction was carried out with the E.Z.N.A. SP Fungal DNA Mini Kit (Omega Bio-Tek, Norcross, GA, USA) and NucleoSpin Plant II Mini Kit (Macherey-Nagel, Düren, Germany) following the instructions of the manufacturers. The ITS (internal transcribed spacer) region of the nrDNA which is the universal fungal DNA barcode region (Schoch et al. 2012) was amplified using the primer pairs ITS1F/ITS4 (White et al. 1990; Gardes and Bruns 1993) as described in Papp and Dima (2018). The primers LR0R (Rehner and Samuels 1994) and LR5 (Vilgalys and Hester 1990) were used to amplify the partial 28S nrRNA gene (LSU) of the nrDNA operon region. The primers EF1-983F and EF1-2218R (Rehner and Buckley 2005) were used to amplify part of the translation elongation factor 1α (tef1-α). Sequencing of the amplicons with the primers used for amplification was carried out by LGC Genomics (Berlin, Germany). The sequences were compiled from electrophoregrams using the Staden software package (Staden et al. 2000) and CodonCode Aligner package (CodonCode Corp., Centerville, Massachusetts, USA). Sequences of each locus (ITS, LSU and tef1-α), together with sequences of respective species downloaded from GenBank mainly based on Jeppson et al. (2017), were aligned separately with the online MAFFT version 7 using the E-INS-i strategy (Katoh and Standley 2013) (Table 1). The alignments were checked and edited in MEGA7 (Kumar et al. 2016) and concatenated to one dataset in SeaView 5 (Gouy et al. 2021). Bayesian Inference (BI) analyses were performed with MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) using a GTR + G substitution model. Four Markov chains were run for 10,000,000 generations, sampling every 1,000 generations with a burn-in value set at 4,000 sampled trees. Maximum Likelihood (ML) phylogenetic analysis was carried out with the raxmlGUI 1.3 implementation (Silvestro and Michalak 2012; Stamatakis 2014). The GTR + G nucleotide substitution model and ML estimation of base frequencies were applied for the partitions. ML bootstrap (BS) analysis with 1,000 replicates was used to test the support of the branches. Tulostoma pulchellum (MJ7833) and T. striatum (Fritz 2010‐2) served as outgroups. Intra- and interspecific genetic differences were calculated by dividing the number of differences (substitutions and/or indels) found in the whole ITS region by the length of the region. Phylogenetic trees were visualised and edited in MEGA 7 (Kumar et al. 2016) and deposited together with the alignments at Figshare repository (10.6084/m9.figshare.24112749). Newly-generated sequences were submitted to GenBank. Studied voucher collections are presented in Table 1.

Table 1.
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Sequences used in this study. Newly-generated sequences are marked in boldface.

Name Strain/Voucher Country ITS LSU TEF References
Tulostoma ahmadii HUP SH-33b, holotype Pakistan KP738712 – – Hussain et al. (2016)
Tulostoma albicans B2092, P.S. Catcheside 1266 Australia – MK278628 – Varga et al. (2019)
Tulostoma albicans Cope, NY, Holotype United States KX576548 – – Jeppson et al. (2017)
Tulostoma beccarianum Finy2 Hungary KU519076 KU519076 KU843959 Jeppson et al. (2017)
Tulostoma beccarianum Herb. Bresadola (S), holotype Italy KX640979 – – Jeppson et al. (2017)
Tulostoma berkeleyi JLH MyCoPortal 6604754 United States MK578704 MK578704 – Unpublished
Tulostoma brumale Finy9 Hungary KU519059 KU519059 KU843944 Jeppson et al. (2017)
Tulostoma calcareum Finy4 Hungary KU519088 KU519088 KU843895 Jeppson et al. (2017)
Tulostoma calcareum MJ6965, holotype Sweden KU519086 KU519086 KU843881 Jeppson et al. (2017)
Tulostoma calongei MJ8773, holotype Spain KU518973 KU518973 KU844000 Jeppson et al. (2017)
Tulostoma caespitosum cf. SNMH9 Slovakia MK907419 MK907419 – Unpublished
Tulostoma caespitosum cf. MJ881114 Spain KU519031 KU519031 KU843978 Jeppson et al. (2017)
Tulostoma caespitosum cf. AH15040 Spain KU519032 KU519032 KU843979 Jeppson et al. (2017)
Tulostoma cretaceum NY737977, holotype United States OR722641 OR722660 – This study
Tulostoma cretaceum cf. 1 Knudsen0107 Russia KU518993 KU518993 KU843988 Jeppson et al. (2017)
Tulostoma cretaceum cf. 2 AH13672 Spain KU518998 KU518998 KU843991 Jeppson et al. (2017)
Tulostoma cretaceum cf. 2 AH3995 Spain KU518999 KU518999 KU843992 Jeppson et al. (2017)
Tulostoma cretaceum cf. 2 MJ6194 Spain KU518997 KU518997 KU843989 Jeppson et al. (2017)
Tulostoma cretaceum cf. 2 MJ9304 Spain KU519000 KU519000 KU843990 Jeppson et al. (2017)
Tulostoma cretaceum cf. 3 FP-2023-05-11-1 Kazakhstan OR722639 OR722658 – This study
Tulostoma cretaceum cf. 3 FP-2023-05-11-4 Kazakhstan OR722640 OR722659 – This study
Tulostoma cretaceum cf. 3 SNMH10 Kazakhstan MK907420 MK907420 – Unpublished
Tulostoma cretaceum cf. MJ3821 Hungary KU518994 KU518994 KU843993 Jeppson et al. (2017)
Tulostoma cyclophorum MJ8862 Hungary KU518985 KU518985 KU843963 Jeppson et al. (2017)
Tulostoma domingueziae MLHC24 (CORD), holotype Argentina HQ667594 HQ667597 – Caffot et al. (2011)
Tulostoma dunense BP112640, holotype Hungary OR722622 OR722648 OR707014 This study
Tulostoma dunense DB-2021-11-21-2 Hungary OR722626 – – This study
Tulostoma dunense FP-2019-12-07 Hungary OR722617 OR722643 OR707009 This study
Tulostoma dunense FP-2020-12-06 Hungary OR722618 OR722644 OR707010 This study
Tulostoma dunense FP-2022-01-02-1 Hungary OR722619 OR722645 OR707011 This study
Tulostoma dunense FP-2021-01-02 Hungary OR722620 OR722646 OR707012 This study
Tulostoma dunense FP-2016-06-05 Hungary OR722621 OR722647 OR707013 This study
Tulostoma dunense FP-2021-02-18 Hungary OR722623 OR722649 OR707015 This study
Tulostoma dunense FP-2015-12-06 Hungary OR722624 OR722650 OR707016 This study
Tulostoma dunense FP-2016-12-11 Hungary OR722625 OR722651 OR707017 This study
Tulostoma dunense MJ6103 (as cf. cretaceum) Hungary KU518995 KU518995 KU843994 Jeppson et al. (2017)
Tulostoma dunense MJ7759 (as cf. cretaceum) Hungary KU518996 KU518996 KU843995 Jeppson et al. (2017)
Tulostoma eckbladii Sivertsen930717, TRH9565, holotype Norway KU519069 KU519069 KU843952 Jeppson et al. (2017)
Tulostoma excentricum BPI 729284, holotype United States KU519055 KU519055 – Jeppson et al. (2017)
Tulostoma fimbriatum Finy8 Hungary KU518968 KU518968 KU843912 Jeppson et al. (2017)
Tulostoma fimbriatum Månsson 991010, epitype Sweden KU518963 KU518963 KU843904 Jeppson et al. (2017)
Tulostoma fulvellum Kabát 970428 Slovakia KU518991 KU518991 KU844001 Jeppson et al. (2017)
Tulostoma giovanellae MJ8706 Spain KU519071 KU519071 KU843954 Jeppson et al. (2017)
Tulostoma grandisporum Finy10 Hungary KU519005 KU519005 KU843922 Jeppson et al. (2017)
Tulostoma grandisporum MJ8907, holotype Hungary KU519003 KU519003 KU843924 Jeppson et al. (2017)
Tulostoma hungaricum BP112641, holotype Hungary OR722630 OR722653 – This study
Tulostoma hungaricum FP-2019-01-23 Hungary OR722627 – – This study
Tulostoma hungaricum FP-2021-02-19 Hungary OR722628 – – This study
Tulostoma hungaricum FP-2022-01-02-2 Hungary OR722629 OR722652 OR707021 This study
Tulostoma kotlabae Brůžek 140918 Czech Republic KU519028 KU519028 KU843977 Jeppson et al. (2017)
Tulostoma kotlabae Kotlaba (PRM 704203), holotype Slovakia KX576544 KX576544 – Jeppson et al. (2017)
Tulostoma cf. kotlabae MJ5996 Hungary KU519016 KU519016 KU843966 Jeppson et al. (2017)
Tulostoma cf. kotlabae Finy1 Hungary KU519017 KU519017 KU843967 Jeppson et al. (2017)
Tulostoma cf. kotlabae MJ7795 Hungary KU519020 KU519020 KU843970 Jeppson et al. (2017)
Tulostoma laceratum NY834492 United States OR722642 OR722661 – This study
Tulostoma lloydii Lahti 201210 Italy KU518990 KU518990 KU843965 Jeppson et al. (2017)
Tulostoma lusitanicum LISU-MGA-8 Portugal KX576542 KX576542 – Jeppson et al. (2017)
Tulostoma lysocephalum Long 9639, holotype United States KU519034 KU519034 – Jeppson et al. (2017)
Tulostoma melanocyclum MJ090418 Hungary KU519106 KU519106 KU843890 Jeppson et al. (2017)
Tulostoma cf. nanum MJ4976 Hungary KU519036 KU519036 KU843968 Jeppson et al. (2017)
Tulostoma niveum MJ7692 Sweden KU519078 KU519078 KU843932 Jeppson et al. (2017)
Tulostoma obesum Cooke 2715, isotype United States KX576541 KX576541 – Jeppson et al. (2017)
Tulostoma obesum MJ8695 Spain KU518986 KU518986 KU843985 Jeppson et al. (2017)
Tulostoma pannonicum MJ7764, holotype Hungary KU519010 KU519010 – Jeppson et al. (2017)
Tulostoma pannonicum MJ7803 Hungary KU519011 KU519011 KU843996 Jeppson et al. (2017)
Tulostoma pseudopulchellum AH 11603, paratype Spain KU519012 KU519012 KU843997 Jeppson et al. (2017)
Tulostoma pseudopulchellum AH 11605, holotype Spain KX513827 KX513827 – Jeppson et al. (2017)
Tulostoma pulchellum MJ7833 Hungary KU518957 KU518957 KU843928 Jeppson et al. (2017)
Tulostoma punctatum BPI 729033, lectotype United States KC333071 KC333071 – Jeppson et al. (2017)
Tulostoma punctatum MJ7472 Slovakia KU518952 KU518952 KU843875 Jeppson et al. (2017)
Tulostoma pygmaeum cf. Brůžek 131207 Slovakia KU519041 KU519041 KU843931 Jeppson et al. (2017)
Tulostoma rufum BPI 704578, holotype United States KU519107 KU519107 – Jeppson et al. (2017)
Tulostoma sacchariolens BP112642, holotype Hungary OR722632 OR722654 OR707020 This study
Tulostoma sacchariolens FP-2019-12-06 Hungary OR722631 – – This study
Tulostoma sacchariolens FP-2021-01-24b Hungary OR722633 – – This study
Tulostoma sacchariolens FP-2021-02-18 Hungary OR722634 OR722655 – This study
Tulostoma shaihuludii BP112643, holotype Hungary OR722637 OR722657 OR707019 This study
Tulostoma shaihuludii FP-2020-12-01 Hungary OR722635 – – This study
Tulostoma shaihuludii FP-2020-12-27 Hungary OR722636 OR722656 OR707018 This study
Tulostoma shaihuludii FP-2017-12-09 Hungary OR722638 – – This study
Tulostoma shaihuludii MJ7762 Hungary KU518979 KU518979 KU843981 Jeppson et al. (2017)
Tulostoma simulans MJ3844 Hungary KU519052 KU519052 KU843941 Jeppson et al. (2017)
Tulostoma sp. 10 MJ3813 Hungary KU519029 KU519029 – Jeppson et al. (2017)
Tulostoma sp. 14 MJ5004 Spain KU519039 KU519039 KU843999 Jeppson et al. (2017)
Tulostoma sp. 20 MJ5015 Spain KU519067 KU519067 KU843950 Jeppson et al. (2017)
Tulostoma sp. 21 AH11698 Spain KX640986 KX640986 – Jeppson et al. (2017)
Tulostoma squamosum Larsson 260-06 France KU519097 KU519097 KU843892 Jeppson et al. (2017)
Tulostoma striatum Fritz 2010-2 Mongolia KU518958 KU518958 KU843929 Jeppson et al. (2017)
Tulostoma submembranaceum AH15132, holotype Mexico KX513826 KX513826 – Jeppson et al. (2017)
Tulostoma submembranaceum cf. MJ9296 Spain KU519014 KU519014 KU843984 Jeppson et al. (2017)
Tulostoma subsquamosum MJ4945 Hungary KU519091 KU519091 KU843899 Jeppson et al. (2017)
Tulostoma verrucosum CCB142 United States MG663293 MG663293 – Unpublished
Tulostoma winterhoffii MJ7761 Hungary KU518976 KU518976 KU843916 Jeppson et al. (2017)
Tulostoma xerophilum Long 9688, BPI 802484, holotype United States KX576549 – – Jeppson et al. (2017)

Results

Phylogenetic analysis

The three-locus molecular phylogenetic analyses of the newly-generated and representative Tulostoma sequences were based on 94 ITS, 76 LSU and 60 tef1-α (Table 1) and 3321 characters. In this study, 26 ITS, 26 LSU and 13 tef1-α sequences were newly gained, including the ITS and LSU sequences of the holotype of Tulostoma cretaceum (Table 1). Phylogenetic trees from ML and BI analyses showed congruent topologies and the sequences representing the four new species proposed here formed strongly-supported clades (MLBS/BIPP = 100%/1.00). The best scoring ML tree is shown in Fig. 2.

Figure 2. 

Maximum Likelihood (RAxML) tree of concatenated nrDNA ITS, nrDNA LSU and tef1-α sequences of representative species of the genus Tulostoma and the four newly-introduced species in the present study. Sequences obtained in this study are shown in bold blue. After the voucher number, the species and the country of origin are shown. Then, the type specimens are indicated. ML bootstrap support values (≥ 70) are shown before slashes and Bayesian posterior probabilities (≥ 0.90) are shown after slashes. Highlighted sections indicate affiliations to the four novel Tulostoma species: T. dunense, T. hungaricum, T. sacchariolens and T. shaihuludii. The illustrations exhibit basidiomata and basidiospore characteristics of the novel species. Tulostoma pulchellum (MJ7833) and T. striatum (Fritz) served as multiple outgroups. The scale bar indicates expected changes per site per branch.

Taxonomy

Tulostoma dunense Finy, Jeppson, L. Albert, Ölvedi, Dima & V. Papp, sp. nov.

MycoBank No: MB 849931
Fig. 3

Holotype

Hungary, Tolna, Németkér, open sandy grassland, 18 Oct 2020, P. Finy, I. Ölvedi, FP-2020-10-18 (BP112640, isotype GB). GenBank: ITS OR722622, LSU OR722648, tef1 OR707014.

Etymology

The epithet refers to the continental, open, bare sandy habitat of this species, similar to coastal dunes.

Description

Spore-sac subglobose, depressed-globose, 10–20 mm. Exoperidium hyphal, encrusting sand only at the base of the spore-sac. Endoperidium tough, chalky white or dirty-dingy white, with age becoming greyish, young basidiomata with velvety surface. Mouth prominent, fibrillose-lacerate, irregular sometimes remains unopened for a long time and splits later due to mechanical pressure (wind or trampling). Socket distantly separated from the stem. Stem 35–80 × 1.5–5 mm, initially white, then ochraceous, with age greyish–blackish, longitudinally furrowed, at the base with a volva and a prominent, easily broken pseudorhiza. Gleba ferruginous to brick-red brown, usually scattered on the surface of the spore-sac. Capillitium brown, 2.5–10 µm in diameter with walls 0.7–2.5 µm in diameter, fragile, breaking up at septal levels in 40–350 µm long segments with rounded, not widened ends, rarely branching. Spores subglobose to oval, 4.6–5.2 × 4.0–4.8 µm (av. 4.4 × 4.9 µm), smooth under LM and SEM.

Habitat and distribution

The psammophilous species Tulostoma dunense known so far only from sandy areas of the Great Hungarian Plain of Hungary. It occurs on both sides of the Danube (Kiskunság, Dél-Mezőföld), where open dunes appear. It mainly grows solitary, deep in the sand in large, open sandy areas to bare spots.

Figure 3. 

Tulostoma dunense: a, c–f FP-2020-10-18 (BP112640, holotype), Németkér b FP-2021-01-02, Tázlár a–c basidiocarps d, e basidiospores f capillitium with basidiospores. Scale bars: 1 μm (d); 10 μm (e); 10 µm (f). Photos: a, b, e, f P. Finy c L. Albert d K. Bóka.

Notes

Tulostoma dunense was previously recorded in Hungary by Hollós (1904), Siller and Vasas (1995), Babos (1999), Siller et al. (2005), Siller et al. (2006) and Rimóczi et al. (2011) under the names of T. volvulatum, T. obesum and T. aff. cretaceum. Hollós (1904) included both T. giovanellae and T. dunense under the name T. volvulatum (nom. rej., Altés et al. (1999)) and recorded it in urban places in the City of Kecskemét (now T. giovanellae) as well as in sand dunes (now the new species, T. dunense). The brownish colour of the capillitium characteristic of specimens from sand dunes and largely absent in those from urban habitats, was considered a result of the maturation process. Later (Hollós 1913) corrected his earlier concept and concluded that some of the synonyms he had listed under T. volvulatum in his publication (Hollós 1904), in fact belonged to a complex of several species. He added illustrations of their different types of capillitia (Hollós 1913: tables 3 and 4) and concluded that his concept of T. volvulatum from the sand dunes was a synonym of T. kansense, a smooth-spored species with brownish capillitium described from North America. The capillitium in the samples from urban habitats clearly showed the undulating inner walls of the capillitium typical of T. giovanellae (Hollós 1913: table 3, fig. 6), although Hollós did not identify them under this name. Some 50 years later, Nagy and Babos (1969) recorded T. giovanellae growing on a pavement at the base of a house wall in Budapest. It matched partly the material cited by Hollós (1904) as T. volvulatum, but was decidedly different from the species growing in the sand dunes. Babos (1999) later came to the same conclusion. Altés et al. (1999) studied the holotype material of T. volvulatum and concluded that it was a synonym of T. giovanellae characterised by ornamented spores. They also studied the holotype material of T. obesum with which they identified European collections with completely smooth spores from steppe habitats and the name T. volvulatum was rejected. Rimóczi et al. (2011) accordingly identified the Hungarian species of the sand dunes as T. obesum. Molecular data (Jeppson et al. 2017) later showed that the Hungarian “T. obesum” was not identical with the American holotype of T. obesum, but was closely related to another American species described as T. cretaceum. It was recovered as T. aff. cretaceum by Jeppson et al. (2017). The T. aff. cretaceum from Hungary belongs to a complex of cryptic species with a strong geographical isolation. The type of T. cretaceum was studied and successfully sequenced by Gube (2009), but the ITS and LSU sequences have remained unpublished. We have kindly received these sequences from Matthias Gube allowing us to include them in the phylogenetic analyses. The phylogenetic analyses showed that the type of T. cretaceum formed a distinct lineage within this complex (Fig. 1), proving that this North American species is different from the European and Asian lookalikes. Therefore, the Hungarian collections are proposed here as a novel species, T. dunense, which is closely related to samples of T. aff. cretaceum collected in Hungary, Kazakhstan and in the Russian Federation as well as in Spain (Fig. 1). The main features to distinguish T. dunense from the other species in the complex are mainly phylogenetic- and geographical-based data. Tulostoma dunense has been a protected species under Hungarian law since 2005, but to date, it has erroneously been treated under various misinterpreted and dubious names, i.e. T. volvulatum, T. obesum and T. aff. cretaceum. The ITS region of T. dunense differs from its closest clade represented by a single sequence (T. cf. cretaceum MJ3821, see Fig. 2) by at least 13 substitution and indel positions, which is a similarity of 98%. This sequence might represent a different species, but further collections need to be studied to clarify its taxonomic status. In contrast, low intraspecific genetic variation was detected in T. dunense (0–4 substitution and indel positions). The ITS and LSU sequences of an old collection identified by Long (www.mycoportal.org) under the name Schizostoma laceratum (NY834492) collected in 1941 in New Mexico, were provided for us by Matthias Gube. Our phylogenetic analyses indicate that this specimen belongs to the T. cretaceum complex as a distinct lineage. On the other hand, the nomenclature of the genus Schizostoma, as well as the species S. laceratum (Fries 1829; Léveillé 1846), seems to be problematic and needs further clarification.

Specimens examined

Hungary, Bács-Kiskun, Ágasegyháza, in open sand, 18 Feb 2021, P. Finy, FP-2021-02-18 (ELTE); Bócsa, in open sand, 7 Dec 2019, P. Finy, FP-2019-12-07 (ELTE); Fülöpháza, 11 Apr 2006, T. Knutsson, T. Gunnarsson, J. Jeppson, M. Jeppson, MJ7759 (GB), Ibidem, in open sand, 5 Jun 2016, P. Finy, FP-2016-06-05 (ELTE); Izsák (Soltszentimre), in open sand, 21 Nov 2019, A. Nagy, B. Dima, DB-2021-11-21-2 (ELTE); Kéleshalom, in open sand, 6 Dec 2015, P. Finy, FP-2015-12-06 (ELTE); Ibidem, in open sand, 2 Jan 2022, P. Finy, I. Ölvedi, FP-2022-01-02-1 (ELTE); Tázlár, in open sand, 11 Dec 2016, P. Finy, FP-2016-12-11 (ELTE); Ibidem, in open sand, 2 Jan 2021, P. Finy, FP-2021-01-02 (ELTE). Pest, Örkény, former military training field, sand steppe vegetation, in open sand, 5 Nov 2001, J. Jeppson, M. Jeppson, MJ6103 (GB), Ibidem, in open sand, 6 Dec 2020, P. Finy, L. Albert, FP-2020-12-06 (ELTE).

Tulostoma hungaricum Finy, Jeppson, L. Albert, Ölvedi & Dima, sp. nov.

MycoBank No: MB 849932
Fig. 4

Holotype

Hungary, Bács-Kiskun, Bócsa, open sandy grassland, on sandy sites with scattered vegetation, near Juniperus communis shrubs 24 Jan 2021, P. Finy, FP-2021-01-24a (BP112641, isotype GB). GenBank: ITS OR722630, LSU OR722653.

Etymology

With reference to Hungary where it was discovered.

Description

Spore-sac subglobose, 3–6 mm. Exoperidium hyphal, heavily encrusting sand grains. Endoperidium white, pitted from adhering sand grains. Mouth small, fibrillose- fimbriate with a small and inconspicuous mouth. Socket inconspicuous. Stem slender, 9–15 × 1–1.5 mm, whitish, not bulbous. Gleba ochraceous brown. Capillitium elastic, 2–6 µm in diameter with walls 0.5–2 µm in diameter and moderate branching. Septa in general not widened. Basidiospores subglobose, 4.9–5.7 × 4.5–5.1 µm (av. 5.2 × 4.8 µm), varied in size, with fine, but visible ornamentation. SEM-photos show low verrucae coalescing in short lines.

Figure 4. 

Tulostoma hungaricum: a, c–g FP-2021-01-24a (BP112641, holotype), Bócsa b FP-2019-11-23, Orgovány a–c basidiocarps d, e basidiospores f, g capillitium with basidiospores. Scale bars: 1 µm (d); 10 µm (e); 20 µm (f, g). Photos: a, b, e–g P. Finy c L. Albert d K. Bóka.

Habitat and distribution

Tulostoma hungaricum occurs in the calcareous, sandy steppe areas, in dry and exposed habitats on bare sand. It has, to date, been found on the sheltered and sun-exposed, extremely warm sandy spots on the south-facing sides of Juniperus communis. So far, it has only been found in few localities of the Kiskunság National Park, Central Hungary.

Notes

Tulostoma hungaricum is the smallest Tulostoma species in Europe. It sometimes shares its habitat with T. pannonicum, another species forming small basidiomata. The latter is, however, easily distinguished on its ochraceous stem, membranous exoperidium and smaller spores. Tulostoma hungaricum is an isolated species belonging to the well-supported Clade 7 according to Jeppson et al. (2017), together with T. submembranaceum from Mexico, T. cf. submembranaceum from Spain and the below-described new species T. sacchariolens. In the ITS region, T. hungaricum differs from its closest species (T. submembranaceum, see Fig. 1) by almost 90 substitution and indel positions, which is a similarity of 87%. Low intraspecific genetic variability was observed in T. hungaricum by a difference of 0–3 substitution and indel positions.

Specimens examined

Hungary, Bács-Kiskun, Kéleshalom, open sandy grassland, near Juniperus communis, 2 Jan 2022, P. Finy, I. Ölvedi, L. Albert, FP-2022-01-02-2 (ELTE); Orgovány, open sandy grassland, near Juniperus communis, 23 Nov 2019, P. Finy, I. Ölvedi, L. Albert, FP-2019-11-23 (ELTE); Ibidem, open sandy grassland, near Juniperus communis, 19 Feb 2021, P. Finy, I. Ölvedi, L. Albert, FP-2021-02-19 (ELTE).

Morphologically examined specimens

Hungary, Bács-Kiskun, Bócsa, open sandy grassland, near Juniperus communis, 3 Dec 2022, P. Finy, I. Ölvedi, L. Albert, FP-2022-12-03 (herb. Finy); Fülöpháza, open sandy grassland, near Juniperus communis, 14 Jan 2023, P. Finy, I. Ölvedi, L. Albert, FP-2023-01-14 (herb. Finy); Pest, Tatárszentgyörgy, open sandy grassland, near Juniperus communis, 17 Dec 2022, I. Ölvedi, OP-2022-12-17 (herb. Ölvedi).

Tulostoma sacchariolens Finy, Jeppson, L. Albert, Ölvedi & Dima, sp. nov.

MycoBank No: MB 849933
Fig. 5

Holotype

Hungary, Bács-Kiskun, Bócsa, open disturbed sandy grassland, in a sand pit, on bare ground, 24 Jan 2021, I. Ölvedi, P. Finy, L. Albert, OP20210124 (BP112642, isotype GB). GenBank: ITS OR722632, LSU OR722654, tef1 OR707020.

Etymology

The epithet refers to its unique sweetish floral smell reminiscent of that of, for example, Hebeloma sacchariolens.

Description

Spore-sac subglobose, often flattened to depressed or hemispherical, 5–9 mm. Exoperidium hyphal, heavily encrusting sand, more persistent at the base of the spore-sac. Endoperidium white or dirty white, pitted from detached sand grains. Mouth delicately fimbriate. Socket conspicuous, forming a thickening on the upper part of the stem. Stem 25–50 × 1.5–2.5 mm, whitish, ornamented with orange to reddish fibrils, with age remarkably blackening, thickening towards the base, bulbous. The mature basidiomata have a pronounced sweet floral smell, reminiscent of Hebeloma sacchariolens Quél. or Freesia flowers. Gleba ferruginous brown. Capillitium 2.5–7 µm in diameter with walls 0.8–2.2 µm in diameter, lumen in general scarce, mostly straight, little branching. Most septa slightly widened. Spores subglobose, 4.6–5.3 × 4.1–5 µm (av. 4.6–4.9 µm), with coarse elongated ornamentation. SEM-photos show developed crests arranged in lines.

Figure 5. 

Tulostoma sacchariolens: a, e FP-2021-02-18, Orgovány b FP-2019-12-06, Bócsa c, d, f OP-2021-01-24 (BP112642, holotype), Bócsa a–c basidiocarps d, e basidiospores f capillitium with basidiospores. Scale bars: 1 µm (d); 10 µm (e); 20 µm (f). Photos: a, b, e, f P. Finy c L. Albert d K. Bóka.

Habitat and distribution

Recorded in calcareous, sandy steppe areas, mostly in sunny open habitats with sparse vegetation, often in trampled or otherwise disturbed places. It is currently known only from a few localities in the sandy areas of the Danube–Tisza interfluves in Central Hungary.

Notes

With its fragrant smell and blackening stem, Tulostoma sacchariolens has a unique combination of characters within the genus, easily separating it from any known Tulostoma species. Tulostoma sacchariolens belongs to Clade 7 according to Jeppson et al. (2017) together with T. cf. submembranaceum (MJ9296, see Fig. 2) from Spain, T. submembranaceum from Mexico and the above-described T. hungaricum. It differs from its sister species (T. cf. submembranaceum) in the ITS region by more than 20 substitution and indel positions, which is a similarity of 96%. The intraspecific genetic variability in the ITS region amongst three sequences of T. sacchariolens was zero (Fig. 1), while the ITS sequence of FP-2019-12-06 had six polymorphic sites.

Specimens examined

Hungary, Bács-Kiskun: Bócsa, open sandy grassland, 6 Dec 2019, P. Finy, FP-2019-12-06 (ELTE); Ibidem, open sandy grassland, 24 Jan 2021, P. Finy, I. Ölvedi, L. Albert, FP-2021-01-24b (ELTE); Orgovány, open sandy grassland, 18 Feb 2021, P. Finy, I. Ölvedi FP-2021-02-18 (ELTE).

Morphologically examined specimens

Hungary, Bács-Kiskun: Bócsa, open sandy grassland, 3 Dec 2022, P. Finy, FP-2022-12-03 (herb. Finy); Fülöpháza, open sandy grassland, 14 Jan 2023, P. Finy, FP-2023-01-14 (herb. Finy); Orgovány, open sandy grassland, 4 Dec 2021, P. Finy, I. Ölvedi, L. Albert, FP-2021-12-04 (herb. Finy); Pest, Örkény, open sandy grassland, 12 Jan 2022, I. Ölvedi, OP-2022-01-12 (herb. Ölvedi); Ibidem, open sandy grassland, 10 Dec 2022, P. Finy, I. Ölvedi, L. Albert, FP-2022-12-10 (herb. Finy).

Tulostoma shaihuludii Finy, Jeppson, L. Albert, Ölvedi, D.G. Knapp & Dima, sp. nov.

MycoBank No: MB 849934
Fig. 6

Holotype

Hungary, Bács-Kiskun, Tázlár, open sandy grassland, 11 Dec 2016, P. Finy, FP-2016-12-11 (BP112643, isotype GB). GenBank: ITS OR722637, LSU OR722657, tef1 OR707019.

Etymology

The epithet refers to its being reminiscent of the sandworm Shai-Hulud of the fictional planet Arrakis from the science fiction novel series Dune by Frank Herbert.

Description

Spore-sac subglobose, often flattened to depressed, 7–18 mm, relatively small compared to the size of the stem. Exoperidium hyphal, encrusting sand at the base of the spore-sac. Endoperidium white or greyish-white, pitted from detached sand grains. Mouth fimbriate, somewhat prominent. Socket developed, slightly separated from the stem. The spore-sac rarely detaches from the stem. Stem 30–70 × 3–6 mm, yellowish-brown to orange brown or reddish-brown, with age darkening, longitudinally furrowed, scaly, often curved, at the base slightly bulbous, with a conspicuous, but fragile pseudorhiza. Gleba ferruginous-cinnamon-brown. Capillitium 3.5–7 µm in diameter with walls 0.3–3.2 µm in diameter, mostly straight, sparsely branched, inner wall often undulating. Septa not or slightly widened. Abundant, thin-walled, septate capillitium hyphae present amongst normal capillitium threads. Basidiospores globose, sometimes flattened, 4.1–5.2 × 3.5–4.7 µm (av. 4.1 × 4.6 µm), finely asperulate, ornamentation not always visible under LM. SEM photos show fine warts arranged in lines forming a dense network.

Figure 6. 

Tulostoma shaihuludii: a FP-2020-12-01-3, Fülöpháza b, d–g FP-2016-12-11 (BP112643, holotype), Tázlár c AL-2021-01-24, Bócsa h FP-2017-12-09, Orgovány a–c basidiocarps d, e basidiospores f, g capillitium with basidiospores h thin-walled, septate capillitium hyphae. Scale bars: 1 µm (d); 10 µm (e); 20 µm (f–h). Photos: a, b, e–h P. Finy c L. Albert d K. Bóka.

Habitat and distribution

Occurs in dry and loose calcareous, open sandy habitats of the Festucetum vaginatae natural grasslands. It mainly grows solitary, deeply rooted in the sand in spots with bare sand. It is currently known only from the sandy areas of Central Hungary.

Notes

Tulostoma shaihuludii is similar in stature to T. fimbriatum and T. winterhoffii, but can be easily distinguished by its habitat (open sand) and its microcharacters, particularly the spore wall ornamentation. It belongs to Clade 2 according to Jeppson et al. (2017) and it forms a sister clade of Tulostoma cf. fimbriatum (MJ8701 as “T. sp2” in Jeppson et al. (2017)) from which it differs in the ITS region by 45 substitution and indel positions, which is a similarity of 93%. The intraspecific genetic variability of T. shaihuludii is low (0–3 substitution and indels positions).

Specimens examined

Hungary, Bács-Kiskun, Fülöpháza, Fülöpházi homokbuckák, sand steppe vegetation, 11 Apr 2006, J. Jeppson, M. Jeppson, MJ7762 (GB); Ibidem, open sandy grassland, 1 Dec 2020, P. Finy, I. Ölvedi, FP-2020-12-01-3 (ELTE); Orgovány, open sandy grassland, 9 Dec 2017, P. Finy, FP-2017-12-09 (ELTE); Pirtó, open sandy grassland, 27 Dec 2020, P. Finy, L. Albert, FP-2020-12-27 (ELTE).

Morphologically examined specimens

Hungary, Bács-Kiskun, Bócsa, open sandy grassland, 7 Dec 2019, P. Finy, FP-2019-12-07 (herb. Finy); Ibidem, open sandy grassland, 24 Jan 2021, P. Finy, L. Albert, I. Ölvedi, FP-2021-01-24 (herb. Finy), AL-2021-01-24 (herb. Albert); Ibidem, open sandy grassland, 4 Dec 2021, P. Finy, I. Ölvedi, FP-2021-12-04 (herb. Finy); Ibidem, open sandy grassland, 3 Dec 2022, P. Finy, FP-2022-12-03 (herb. Finy); Fülöpháza, open sandy grassland, 2 Dec 2018, P. Finy, FP-2018-12-02 (herb. Finy); Ibidem, open sandy grassland, 16 Jan 2022, P. Finy, I. Ölvedi, FP-2022-01-16 (herb. Finy); Izsák (Soltszentimre), open sandy grassland, 4 Feb 2016, P. Finy, FP-2016-02-04 (herb. Finy); Ibidem, open sandy grassland, 14 Dec 2016, P. Finy, FP-2016-12-14 (herb. Finy); Ibidem, open sandy grassland, 17 Jan 2019, P. Finy, FP-2019-01-17 (herb. Finy); Ibidem, open sandy grassland, 16 Dec 2020, P. Finy, FP-2020-12-16-1 (herb. Finy); Kéleshalom, open sandy grassland, 6 Dec 2015, P. Finy, FP20151206 (herb. Finy); Ibidem, open sandy grassland, 2 Jan 2022, P. Finy, I. Ölvedi, FP-2022-01-02-3 (herb. Finy); Kiskunhalas, open sandy grassland, 22 Dec 2019, P. Finy, FP-2019-12-22 (herb. Finy); Ibidem, open sandy grassland, 5 Jan 2023, P. Finy, I. Ölvedi, FP-2023-01-05 (herb. Finy); Kunbaracs, open sandy grassland, 5 Feb 2022, P. Finy, I. Ölvedi, FP-2022-02-05 (herb. Finy); Orgovány, open sandy grassland, 13 Aug 2017, P. Finy, FP-2017-08-13 (herb. Finy); Ibidem, open sandy grassland, 18 Feb 2021, P. Finy, I. Ölvedi, FP-2021-02-18 (herb. Finy); Pirtó, open sandy grassland, 16 Jan 2016, P. Finy, FP-2016-01-16 (herb. Finy); Tázlár, open sandy grassland, 11 Dec 2016, P. Finy, FP-2016-12-11 (herb. Finy); Pest, Örkény, open sandy grassland, 12 Jan 2022, I. Ölvedi, OP-2022-01-12 (herb. Ölvedi); Tatárszentgyörgy, open sandy grassland, 1 Jan 2022, I. Ölvedi, OP-2022-01-01 (herb. Ölvedi); Ibidem, open sandy grassland, 10 Dec 2022, P. Finy, FP-2022-12-10 (herb. Finy); Ibidem, open sandy grassland, 17 Dec 2022, I. Ölvedi, OP-2022-12-17 (herb. Ölvedi); Tolna, Paks, open sandy grassland, 4 Feb 2018, P. Finy, FP-2018-02-04 (herb. Finy); Ibidem, open sandy grassland, 22 Jan 2021, P. Finy, FP-2021-01-22 (herb. Finy); Ibidem, open sandy grassland, 9 Jan 2022, I. Ölvedi, P. Finy, OP-2022-01-09 (herb. Ölvedi); Ibidem, open sandy grassland, 27 Feb 2022, P. Finy, FP-2022-02-27 (herb. Finy).

Discussion

The results of our study further emphasise the high species diversity amongst the stalked puffballs (Tulostoma) in East Central Europe, as previously indicated by Jeppson et al. (2017). In Hungary, so far 19 species have been recorded, including the four new species proposed in this study. The Pannonian, dry and sandy grasslands between the rivers Danube and Tisza, as well as adjacent areas in Central Hungary, harbour to date 66% of all described species of Tulostoma known to occur in Europe (29 spp.). The dry, sandy grasslands in Central Hungary have a long continuity as natural grasslands or as sheep pastures and are characterised by steppe flora and fauna. Both natural and grazed habitats are rich in gasteroid fungi, but usually their species composition is different. The summer and autumn temperatures in the sand are extremely high and the yearly precipitation is low. The dry and drought-resisting basidiomata of Tulostoma species could be considered as adaptations to xeric conditions. The development of the basidiomata occurs mainly in late autumn and early winter.

Tulostoma species are generally rare (although locally abundant) and the current knowledge of their population structures in Europe is limited. However, their occurrences are highly dependent on the habitat status where they grow and changes in land management are likely to be detrimental to their populations. A vast majority of the European Tulostoma species are Red-Listed in the countries where they occur (http://www.eccf.eu/redlists-en.ehtml).

In addition to the four novel species proposed herein, the results from previous works (e.g. Jeppson et al. (2017)) and our ongoing studies indicate the presence of many more undescribed species of Tulostoma in Central Europe.

Acknowledgements

The work was supported by the ÚNKP-22-5 New National Excellence Program of the Ministry for Culture and Innovation from the source of the National Research, Development and Innovation Fund, the National Research, Development and Innovation Office of Hungary (FK-143061, the ELTE Institutional Excellence Program 2020 (TKP2020-IKA-05), Diagnostics and Therapy 2). Bálint Dima is grateful to the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. We thank to Csilla Gergely for her assistance in laboratory work. We are grateful to Matthias Gube (Germany) for allowing us to publish the type sequences of Tulostoma cretaceum and the sequences of Schizostoma laceratum.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

New National Excellence Program of the Ministry for Culture and Innovation from the source of the National Research, Development and Innovation Fund (Hungary); National Research, Development and Innovation Office (Hungary); János Bolyai Research Scholarship of the Hungarian Academy of Sciences.

Author contributions

Conceptualisation: PF, BD. Methodology: PF, MJ, DGK, VP, KB, DV, BD. Validation: PF, MJ, LA, IÖ. Formal analysis: PF, MJ, DGK, DV, BD. Investigation: PF, MJ, LA, IÖ, KB, BD. Resources: PF, MJ, LA, IÖ, BD. Data Curation: PF, MJ, DGK, LA, IÖ, DV, BD. Writing - Original draft: PF, MJ, VP, BD. Writing - Review and Editing: PF, MJ, DGK, VP, LA, IÖ, KB, DV, GMK, BD. Visualisation: PF, DGK, VP, LA, IÖ, KB. Supervision: MJ, GMK, BD. Funding Acquisition: GMK, BD.

Author ORCIDs

Dániel G. Knapp https://orcid.org/0000-0002-7568-238X

Viktor Papp https://orcid.org/0000-0001-6994-8156

Károly Bóka https://orcid.org/0000-0002-1324-3592

Gábor M. Kovács https://orcid.org/0000-0001-9509-4270

Bálint Dima https://orcid.org/0000-0003-2099-3903

Data availability

All the data that support the findings of this study are available in the main text or in publicly accessible data repositories, as indicated in the text.

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