Introduction

The globally distributed genus Amanita Pers. is one of the iconic groups of basidiomycetous macrofungi in Agaricales. The genus includes economically important edible species as well as lethally poisonous ones. A supposedly strictly saprotrophic basal lineage of Amanita with squarrose universal veil was isolated in a separate genus by Vizzini et al. (2012) as Aspidella, E. J. Gilbert (nom. illegit.) and then by Redhead et al. (2016), as Saproamanita Redhead, Vizzini, Drehmel & Contu. The splitting of Amanita into two genera along the lines of trophic status did not receive broad acceptance in the scientific community (Tulloss et al. 2016). Apart from Saproamanita species, the genus Amanita primarily includes a majority of ectomycorrhizal (EcM) species, representing functionally important elements in most terrestrial ecosystems, from xerophilic tropical forests to arctic-alpine micro-sylvae (Gilbert 1941; Bas 1969; Singer 1986). In some either natural or planted forests, with native as well as introduced trees, some species of Amanita may dominate the local EcM communities and possibly address toxicological issues (e.g. Wolfe et al. 2010).

Few studies during the past few decades have explored different aspects of the phylogeny of the genus (Weiß et al. 1998; Drehmel et al. 1999; Oda et al. 1999; Zhang et al. 2004; Redhead et al. 2016; Tulloss et al. 2016; Cui et al. 2018). The systematic proposed by Cui et al. (2018), which divides Amanita into three subgenera (Amanita, Amanitina and Lepidella) and eleven sections based on morphological and molecular data, is now the most widely accepted one. The subgenus Amanita includes four sections (Amanita, Amarrendiae, Caesareae and Vaginatae) whereas the sections Amidella, Arenariae, Phalloideae, Roanokenses, Strobiliformes and Validae are members of the subgenus Amanitina. The subgenus Lepidella which basically represents the genus Saproamanita accepted by some authors (e.g. Redhead et al. 2016) includes the solely section Lepidella. The number of species of the genus Amanita is estimated to be around 1000 (Yang et al. 2018), of which approximately 700 are accepted worldwide (Codjia et al. 2023). In the last two decades, about 220 taxa of the genus Amanita have been published as new to science from all over the world. This can be explained both by the examination of samples from previously less studied areas and by the increasing use of molecular methods that contributed to the discovery of a significant number of new taxa, even from existing collections. As a result, a high number of morphologically defined species are now found to be species complexes (Zhang et al. 2015, Thongbai et al. 2018).

The genus has extensively been studied in Europe by means of morphological approaches (e.g. Sartory and Maire 1923, Gilbert 1941, Bas 1969, Romagnesi 1992, Contu 1997, 1999, 2003, Traverso 1998, Neville and Poumarat 2009). Molecular phylogenetic research involving European species of the genus date back to the end of the twentieth century, and the genus has recently received increasing attention (Weiß et al. 1998; Moreno et al. 2008; Redhead et al. 2016, Vizzini et al. 2016; Loizides et al. 2018, Hanss and Moreau 2020, Alvarado et al. 2022).

Amanita sect. Vaginatae (Fr.) Quél., so-called ‘ringless Amanitas’, is one of the taxonomically most challenging and most diverse groups within the genus with ca. 400 estimated number of species (Ševčíková et al. 2021). Morphologically, the species of this section can be characterised by the striate-sulcate pileus margin, the lack of annulus, the elongate stipe without having a bulb and the presence of more or less well-developed universal veil remnants at the stipe base as well as the clampless basidia (Yang 1997). Recent molecular phylogenetic studies revealed a globally high species diversity of this section (e.g. Kim et al. 2013; Malysheva & Kovalenko 2015; Tang et al. 2015; Vizzini et al. 2016; Liu et al. 2017; Cui et al. 2018; Thongbai et al. 2018; Lambert et al. 2018; Ullah et al. 2019). Hanss and Moreau (2020) published the first comprehensive revision of the European species in Amanita sect. Vaginatae focusing on the grey-coloured species. They recognised and fixed 28 phylogenetic species based on nrDNA ITS sequences and morphological data. After this fundamental work, further new Vaginatae species and records have been published from Europe in various taxonomic papers (Ševčíková et al. 2021; Migliozzi and Donato 2022a, b; Illescas and Plaza 2022; Plaza 2022; Hanss and Moreau 2022). During our revision based on phylogenetic and morphological data of the sect. Vaginatae in Hungary, we discovered several lineages which were not possible to assign to currently recognized taxa (Varga et al. 2021). Extending our study on a European scale made it possible to compare our data with those of other European regions such as Western and Northern Europe. These comparisons reinforced that those lineages are distinct from known species. As a result, here, we describe three new species of Amanita sect. Vaginatae based on phylogenetic evidence of 4-locus (nrDNA ITS, nrDNA LSU, RPB1 and TEF1-α) molecular phylogenetic analysis, macroscopic and microscopic data. Basidiospore size and shape in Amanita sect. Vaginatae have been known as mainly overemphasised characters in species delimitation in the past; however, in some species it can still be a stable character (Vizzini et al. 2016). A statistical analysis on basidiospore measurements was systematically driven for the species studied here to test whether basidiospore biometrics would be suitable characters to delimit species among the studied species of sect. Vaginatae. Our results contribute to further insights of the diversity of ringless Amanitas in Europe as well as provide the first comprehensive revision of this group in Hungary.

Materials and methods

Macromorphological studies

A total of 151 specimens of Amanita sect. Vaginatae and four specimens of other sections of the genus were collected from Hungary, France, Austria, Italy, Norway and Romania. Macroscopic observations were made based on fresh collections. All collections were photographed in situ or in laboratory on plate in order to document the main morphological appearance of the basidiomata. Photos were taken in situ on the field or in laboratory. The shape of the volva and their terminology were adopted from Fraiture (1993) (Fig. 1). Macroscopic descriptions given in this study avoid superfluous details of macroscopic characters that obscure the understanding of the unambiguous features of the species. In the descriptions, we focused on highlighting the particularities of the new species introduced in this study.

Fig. 1
figure 1

Types of the shapes of volva in Amanita sect. Vaginatae based on Fraiture (1993): I universal veil friable, either powdery (a) or forming a fragmented volva at the base of the stipes (b); II lower part close to the stipe, upper part well away from the stipe; III vaginate volva; the base may be variable, more or less long attached to the stipe or slightly dilated and IV saccate volva, thick to very thick, generally markedly membranous

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Microscopy

The principles and methods used for microscopy as well as terminology used for microscopical structures and spore features follow Bas (1969). The types of subhymenium follow Bas (1969) and were reproduced and numbered from 1 to 5 in Hanss and Moreau (2020). Nikon Eclipse 220 and Carl Zeiss 4013014 light microscopes with magnifications of 100×, 400× and 1000× were used to examine the microscopic features. Preparations for microscopy were obtained from dried material. Sections made with a Ranvier microtome or otherwise extracted fragments were reinflated in “ramollisseur GDS” and mounted in SDS Congo red (after Clémençon 1999) or alternatively reinflated in 3% KOH and observed in aqueous Congo red or in 3% KOH, or alternatively reinflated and observed in ammoniacal Congo red with phloxin. Photographs were taken by Canon EOS 700D and Nikon Coolpix P5100 cameras. Spore length, width and Q-value (length/width ratio of a basidiospore in side-view) were calculated using Piximètre (www.piximetre.fr) or manually determined for 30 spores per specimen. The data were analysed by ANOVA and other tests and post hoc Tukey’s pairwise tests were conducted to study differences of spore data of different species using the software PAST4.09 (Hammer et al. 2001). Sections of volva were made on their upper part, where remains of partial veil on their internal face are usually absent.

Molecular analyses

Genomic DNA was extracted from 4–5 mm pieces of dried herbarium specimens using (i) the NucleoSpin Plant II Mini Kit (Macherey-Nagel, Düren, Germany) following the manufacturer’s instructions (in ELTE, Budapest, Hungary) or (ii) based on the protocol communicated by the MycoSeq project of the Société Mycologique de France/CEFE Montpellier (see also Hanss and Moreau 2020) using the REDExtract-N-Amp Plant PCR Kit (Sigma-Aldrich, Saint Louis, MO, USA), also following the manufacturer’s instructions, or by Alvalab (Spain) employing a modified protocol based on Murray andThompson (1980).

We targeted four nuclear loci: the nuclear ribosomal internal transcribed spacer regions (nrDNA ITS), nuclear ribosomal large subunit (nrDNA LSU), DNA-dependent RNA polymerase II largest subunit (RPB1) and the translation elongation factor 1-alpha (TEF1-α) genes.

The ITS region was amplified by PCR with the combinations of primers ITS1F, ITS5, ITS4, ITS4B and, for problematic cases, ITS2 and ITS3 (White et al. 1990; Gardes & Bruns 1993), under the conditions detailed in Richard et al. (2015) or used the following PCR protocol: initial denaturation step for 5 min at 94 °C, followed by 35 cycles of denaturation at 94 °C, for 30 s, annealing at 52 °C for 30 s and extension at 72 °C for 40 s and final extension at 72 °C for 10 min. The primer pairs LR0R/LR7 (Vilgalys and Hester 1990) for LSU, RPB1-Af/RPB1Cr (Matheny et al. 2002) for RPB1 and EF1-983F/EF1-2218R (Rehner and Buckley 2005) for TEF1-α were used for rest of the PCR amplifications which were performed in Tianlong Genesy 96T Thermal Cycler in 25-μl reaction mixture (also used for most of the ITS PCR) contained the following amounts 14.1 μl ddH2O, 2.5 μl 10× Dream Taq Buffer (Thermo Scientific), 2.5 μl 2mM dNTP (Thermo Scientific), 1.25 μl of each primer (10 μM), 0.4 μl Dream Taq DNA polymerase (Thermo Scientific, 5 U/µl) and 3 μl DNA extract.

Touchdown PCR was used to amplify RPB1, which differs from the previous one in 6 preceding repetitive cycles in which the annealing temperature of 52 °C is reached gradually from 58 °C by decreasing 1 °C per cycle. Unlike the program used for ITS, annealing at 59 °C for 30 s and extension at 72 °C for 1 min, settings were used to amplify TEF1-α region.

In some cases, ITS amplification was made directly from basidiome samples by the Phire Plant Direct PCR Kit (Thermo Scientific, USA). This 20-μl-PCR mixture contained 7 μl ddH2O, 10 μl 2× Phire Plant Direct PCR Master Mix, 1 μl of each primer (10 μl) and 1 μl template DNA. The reaction included 5 min initial denaturation step at 98 °C, 41 cycles of denaturation at 98 °C, for 5 s, annealing at 55 °C for 5 s and extension at 72 °C for 7 s and final extension at 72 °C for 1 min. PCR products were checked in 1.5% agarose gel stained with EcoSafe, and illuminated by UV-transilluminator (NuGenius, Syngene).

Sanger sequencing was performed with primers used in PCR at LGC Genomics (Berlin, Germany) or at Eurofins Genomics (Ebersberg, Germany). PreGap4 and Gap4 of Staden Package (Staden et al. 2000) as well as the CodonCode Aligner package (CodonCode Corp., Centerville, Massachusetts, USA) were used for sequence assembly and editing. The purified sequences were compared to those downloaded in GenBank (https://www.ncbi.nlm.nih.gov/genbank/) and UNITE (https://unite.ut.ee/) databases using BLASTn (Altschul et al. 1990). ITS, LSU, RPB1 and TEF1-α sequences obtained were aligned for each gene separately with MAFFT online v7 (http://mafft.cbrc.jp/alignment/server) using E-INS-i strategy for ITS, G-INS-i for LSU, FFT-INS-i for RPB1 and TEF1-α (Katoh and Standley 2013). In the case of ITS, the phylogenetically informative indels were coded using the “simple indel coding” algorithm (Simmons et al. 2001) in FastGap 1.2 (Borchsenius 2009). Nucleotide alignments and the binary character matrix were edited in MEGA 7 software (Kumar et al. 2016) and concatenated in SeaView 5 (Gouy et al. 2021).

For maximum likelihood phylogenetic analysis, raxmlGUI 2.0 (Edler et al. 2021) was employed. The GTRGAMMA substitution model was applied for the six nucleotide partitions (ITS1, 5.8S, ITS2, LSU, RPB1 and TEF1-α), while the one binary partition (indel characters) was set to default. To determine support value, a 1000-replicant rapid-bootstrap analysis was chosen. MrBayes 3.2.6 (Ronquist et al. 2012) was used to infer Bayesian phylogeny. The alignment was partitioned as describe above. For the six nucleotide partitions the GTR + Γ substitution model was applied, while the two-parameter Markov model was set for the binary characters. Two independent runs of four Markov Chain Monte Carlo (MCMC) were performed each for 10 million generations, sampling every 1000th generation. The first 30% of the trees was discarded as burn-in. For the remaining trees, a 50% majority rule consensus phylogram with posterior probabilities as nodal supports was computed. The best scoring ML tree from Maximum Likelihood analysis was further edited in MEGA 7 and Microsoft PowerPoint.

Results

Phylogeny

The final alignment is composed of 390 nrDNA ITS, 201 LSU, 75 RPB1 and 93 TEF1-α sequences and 5453 characters (ITS: 892, LSU: 1441, RPB1: 1367, TEF1-α: 1187 and binary characters: 566). The concatenated 4-locus alignment is included in the Supplementary Material. Phylogenetic trees from maximum likelihood and Bayesian inference analyses showed congruent topologies. The best-scoring ML tree is chosen to depict phylogenetic relationships in Amanita sect. Vaginatae shown in Figs. 2 and 3. The 370 newly generated sequences (157 ITS, 84 LSU, 74 RPB1 and 55 TEF1-α) were deposited in GenBank (Table 1).

Fig. 2
figure 2

Phylogenetic tree based on maximum likelihood and Bayesian analyses, derived from 4-locus dataset (ITS, LSU, RPB1 and TEF1-α) showing relationships above species level within Amanita sect. Vaginatae using sect. Amanita and sect. Phalloideae as outgroups. Stirp level clades are compressed. Corrugation shows previously recognized stirpes (Hanss and Moreau 2020), while black clades indicate newly named stirps in this study. ML bootstrap values (≥ 70%) and Bayesian posterior probabilities (≥ 0.8) are reported above branches, on the left and right sides of the slashes, respectively. The scale bar represents 0.03 expected change per site per branch

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Fig. 3
figure 3figure 3figure 3figure 3

Phylogenetic tree based on maximum likelihood and Bayesian analyses, derived from 4-locus dataset (ITS, LSU, RPB1 and TEF1-α) of Amanita sect. Vaginatae specimens and those of reference materials. The sequences newly generated in this study are in bold. ML bootstrap values (≥ 70%) and Bayesian posterior probabilities (≥ 0.8) are reported above branches, on the left and right sides of the slashes, respectively. The scale bar represents 0.03 expected change per site per branch

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Table 1 Sequences used in this study
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A compressed tree is shown in Fig. 2 presenting six clades and 22 stirps as the main phylogenetic structure within sect. Vaginatae. Half of the lineages (stirps Arctica, Arenicola, Basiana, Crocea, Friabilis, Lignitincta, Nivalis, Spadicea, Verrucosivolva, Vladimirii and Zonata) are newly recognized. Most of the lineages received strong support in both ML and BI analyses, especially those of containing European species. Some non-European species could not be assigned any of these stirps under the current sampling; therefore, they remained singletons. Fig. 3 represents the species level phylogenetic relationships of sect. Vaginatae. We recognized 36 species (Figs. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14) from our studied 151 European specimens which clustered in 17 stirps across the phylogenetic tree (Fig. 3). We could not assign any proper scientific name to four species level lineages, of which in one case we applied the ‘aff.’ prefix (A. aff. arctica) using morphological similarity and leaving taxonomic conclusions to later studies. The remaining three distinct species of which two belong to stirps Fulva while one is a member of the stirps Coryli are described here new to science based on molecular and morphological evidences.

Fig. 4
figure 4

Representative specimens of Amanita deflexa: a, b JMH2019039 (LIP 0004489, holotype, France); c, d DB-2020-10-03-1 (AD52, Hungary). Photos: a, b J-L. Muller; c, d B. Dima

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Fig. 5
figure 5

Important microstructures of the newly described Amanita deflexa: a external layer of the volva (JMH2019029), b main layer of the volva (JMH2019029); c basidiospores (DB-2020-10-03-1), d subhymenial cell (JMH2019029). Photos: a–b, d J-M. Hanss, c D. Varga. Scale bars: a = 20 µm, b = 75 µm, c = 20 µm, d = 20 µm

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Fig. 6
figure 6

Representative specimens of Amanita griseofulva: a PAM21081109 (LIP 0404490, holotype, France), b DB-2020-07-05-6 (AD37, Hungary), c DB-2020-07-25-2 (AD09, Hungary), d DB-2020-06-27-4 (AD14, Hungary), e (PAM16091803, France); and Amanita griseofulva f. albida: f DB-2021-07-31-2 (AD115, holotype, Hungary). Photos: a, e P-A. Moreau; b, c, d B. Dima; f A. Nagy

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Fig. 7
figure 7

Important microstructures of the newly described Amanita griseofulva: a structure of the volva (PAM20181109), b subhymenium (PAM20181109), c basidiospores (DB-2020-07-05-6), d lamellar trama (PAM20181109). Photos: a–b, d J.-M. Hanss, c D. Varga. Scale bars: a = 65 µm, b = 15 µm, c = 20 µm, d = 100 µm

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Fig. 8
figure 8

Representative specimens of Amanita opaca: a AB11-08-60 (LIP 0004500, holotype, France), b AB12-05-05 (France), c JMH2020007 (France), d DB-2020-06-28-8 (AD86, Hungary), e, f DB-2021-06-19-5 (AD104, Hungary), g DB-2021-06-19-3 (AD102, Hungary); and A. opaca f. cettoi: h JMH2016027 (LIP 0004499, holotype, France), i DB-2020-08-02-3 (AD39, Hungary). Photos: a, b A. Bidaud; c, h J.-M. Hanss; d, e, f, g, i B. Dima

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Fig. 9
figure 9

Important microstructures of the newly described Amanita opaca: a pileipellis and context (JMH2020007), b twisted hyphae of volva enveloping physaloid cells (JMH2020007), c basidiospores (DB-2021-06-19-2), d subhymenial cell (JMH2020007). Photos: a–b, d J.-M. Hanss, c D. Varga. Scale bars: a = 120 µm, b = 80 µm, c = 20 µm, d = 10 µm

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Fig. 10
figure 10

Representative species of Amanita sect. Vaginatae in Europe. Stirps Mairei: a A. brunneofuliginea f. ochraceopallida DB-2021-08-07-4 (AD129, Norway), b A. supravolvata DB-2022-10-17-1 (AD157, Hungary); stirps Albogrisescens: c A. alseides DB-2020-06-28-3 (AD13, Hungary), d A. albogrisescens DB-2022-10-21-2 (AD152, Hungary); stirps Simulans: e A. simulans BoGy-2019-05-28 (AD19, Romania), f A. beckeri JMH2015027 (France), g A. beckeri GC17092307 (France). Photos: a, b, d B. Dima; c A. Nagy; e Gy. Bodó; f J.-M. Hanss; g G. Corriol

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Fig. 11
figure 11

Representative species of Amanita sect. Vaginate in Europe. Stirps Lividopallescens: a A. dryophila AL-2020-08-21-1 (AD38, Hungary), b A. lividopallescens DB-2018-06-12-1 (AD76, Hungary), c A. sublividopallescens DB5062 (AD94, Hungary); stirps Betulae: d A. betulae DB-2021-08-08-2 (AD127, Norway); stirps Coryli: e A. prudens DB-2020-07-05-2 (AD18, Hungary), f A. prudens AB10-08-100 (France), g A. coryli DB-2020-07-05-7 (AD03, Hungary); stirps Vladimirii: h A. vladimirii DB-2022-09-24-1 (AD166, Austria). Photos: a L. Albert; b I. Fedor; c, d, e, g, h B. Dima; f A. Bidaud

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Fig. 12
figure 12

Representative species of Amanita sect. Vaginatae in Europe. Stirps Crocea: a A. crocea DB-2021-08-08-3 (AD122, Norway), b A. crocea PG-2023-07-27-1 (A3108, Hungary), c A. subnudipes DB-2022-10-02-3 (AD158, Hungary), d A. subnudipes DB-2023-06-10-5 (A3024, Hungary – sequenced, but not included in the analyses); stirps Submembranacea: e A. submembranacea DB-2018-07-31-1 (AD25, Italy), f A. mortenii DB-2021-08-27-1 (AD112, Austria); stirps Arctica: g A. aff. arctica DB-2021-08-12-1 (AD114, Norway); stirps Friabilis: h A. friabilis AL13/109 (A3093, Hungary – sequenced, but not included in the analyses), i A. suberis DB-2022-10-02-1 (AD149, Hungary); stirps Ceciliae: j A. ceciliae DB-2018-07-08-5 (AD22); Photos: a, d A. Nagy; b, c, e, g, i, j B. Dima; f, h L. Albert

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Fig. 13
figure 13

Representative species of Amanita sect. Vaginatae in Europe. Stirps Fulva: a A. fulva DB-2020-06-13 (AD42, Hungary), b A. fulva JMH2019003 (France), c A. fulvoides DB6704 (AD66, Hungary), d A. fulvoides DB-2018-05-31-2 (AD68, Hungary), e A. umbrinolutea DB-2021-08-27-2 (AD116, Austria); stirps Argentea: f A. huijsmanii NA-0010 (AD47, Hungary), g A. electra DL140901 (holotype, France); stirps Spadicea: h A. spadicea DB-2022-10-09-1 (AD167, Hungary). Photos: a A. Nagy; b J.-M. Hanss; c, d, e, h B. Dima; f A. Nagy; g D. Lucas

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Fig. 14
figure 14

Representative species of Amanita sect. Vaginatae in Europe. stirps Nivalis: a A. sponsa PAM18102001 (holotype, France), b A. sponsa (PAM18110303, France), c A. griseocaerulea (DL141120A, France) d A. griseocaerulea DL2009071 (France), e A. nivalis DB-2021-08-12-2 (AD128, Norway); stirps Magnivolvata: f A. calida DB-2023-06-23-2 (A3051, Hungary — sequenced, but not included in the analyses), g A. calida DB-2020-07-19-17 (AD32, Hungary), h A. herculis DB-2022-10-21-3 (AD150, Hungary), i A. battarrae DB4685 (AD92, Hungary). Photos: a, b P.-A. Moreau; c, d D. Lucas, e, g, h, i B. Dima; f K. Császárné-Erdélyi

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Taxonomy

Amanita deflexa Hanss, Dima & L. Albert, sp. nov., Figs. 4, 5.

MycoBank: MB#846787.

Holotype: France, Haut-Rhin, la Gauchmatt, Soulzmatt, in a Pinus nigra forest, J-L. Muller, 1 Sept 2014, LIP 0004489 (isotype JMH2019039 in herb. J-M. Hanss); GenBank PP375656 (ITS) and PP273643 (TEF1-α).

Etymology: the epithet deflexus, participle of deflecto, here granted to the feminine with Amanita, the verb deflecto being taken in the acceptance to turn away, to move away.

Diagnosis: Differs from Amanita fulva by the pileus colour that varies with the age of the fungus, by the volva which is strongly coloured inside and on the outer upper rim, by the two-layered volva, as well as by nucleotide composition of ITS marker.

Description: Pileus campanulate to convex plan and then slightly depressed with a small nipple with age, 4-cm diameter, with ridged margin. The colour is very dark at first and then it takes a shade varying from yellow to orange, with the centre much darker. With aging, it becomes dull. Lamellae free, crowded, whitish to cream. Stipes 5–10 cm long, smooth, long white, which can take with age faintly coloured reflections in the tones of the pileus. Volva 2.5–3 cm high, type II, externally white, tinted with an intense colour varying from red-brown to yellow on the upper part, near the edge and inside. Basidiospores 7–12.5 × 7–11.7 µm, globose to subglobose, Lav = 11.02 µm, Wav = 10.35 µm, Qav = 1.068. Mature basidia few, rather long, 60–80 × 20–25 µm, sometimes curved at the base, sterigmata 5–6 µm long. Lamellar trama up to 100-µm thick formed by a mediostratum of hyphae 3–4-µm thick mixed with elongated oval physaloid cells forming chains, e.g. 60 × 20 µm or 50 × 14 µm. Few oval physaloid cells of the same sizes presented sometimes under the subhymenium. The whole forms a compact matrix. Subhymenial cell under the mature basidia ramose, type 1a, enlarged at the top and markedly attenuated at the base, 15–20 × 6–8 µm, more rarely cylindrical. Subhymenial cells under young basidia ramose to strongly inflated ramose. The other elements of the subhymenial tree are of various shapes, up to subcellular. Lamellar edge sterile by the presence of numerous marginal cells with largely oval head (30 × 20 µm to 40 × 30 µm), more rarely subglobose (30 × 30 µm), immersed in a gelatinous matrix but occasionally can more or less emerge in places. The peduncle is very variable in length as in all cells of this type. Pileipellis about 200-µm thick, ixocutis with 25-µm-wide gelatinous matrix, containing 2-µm-thick congophobic parallel hyphae, stratum of the parallel hyphae 3–4(–6)-μm-thick, elongated to cylindrical, ellipsoid acrophysalydes, e.g. 70 × 20 μm present, but 160 × 22 µm becoming more numerous towards the context. Differentiated subpellis lacking. Context mainly composed of elongated acrophysalydes, e.g. 150 × 50 µm to 200 × 55 µm, mixed with hyphae of 5–6 µm, is oriented in all directions. The whole forms a medium-intricate and weakly resistant tissue. Volva thin, with a thickness of 500–600 µm comprising a weakly gelified, filamentous, 50-µm thick outer stratum and a main stratum composed of tangled hyphae mixed with physaloid cells. The outer stratum is weakly gelified on its outer part. It is composed of 3-µm-thick septate hyphae, parallel to the stipe, with quite rare and clearly elongated physaloid cells. The main stratum consists of 5–7(–10)-µm-thick hyphae, septate, tangled in all directions, clearly entangled outwards and entangled much looser inwards. The physaloid cells, in average proportion, are equally distributed over thickness, subspherical (e.g. 50 × 40 µm or 70 × 60 µm), oval and terminal (e.g. 60 × 35 µm), cylindrical (e.g. 90 × 20 µm), rarely elongated, sometimes with 3 connections (e.g. 90 × 20 µm).

Phylogenetic relationship: Amanita deflexa is a sister species of the recently typified A. fulva (Moreau et al. 2023), from which it differs by 5 substitution and indel positions (99% similarity) in the ITS region and by 8 nucleotides (98% similarity) in the TEF1-α region. The A. fulva complex is a challenging group with several closely related but well-separated species, distributed globally (Fig. 3).

Habitat and distribution: Associated with Pinus sylvestris on acidic soil and with P. nigra on calcareous soil. Distribution is currently uncertain due to the confusion with A. fulva. Currently known from Europe (France and Hungary — own collections and one GenBank entry: MH508370) and USA (Pennsylvania, Indiana, Massachusetts and Virginia according to ITS sequence data from GenBank entries: JX860436, KP284293, MK522007, MK522008, MZ668288, ON392627, ON243897 and OR026696).

Comments: this taxon is recognizable by its reminiscence to the habitus of Amanita fulva, by the variable colour of the pileus, by the particular colour of the inner and the upper outer surfaces of the volva. The main characteristic of microscopic differentiation is the presence of an external stratum 50-µm-thick, partially gelified, occurring parallel to the stipe hyphae. It is the only European taxon of the stirps Fulva to have a two-layered volva. The subhymenial cells are of the same type as those of Amanita fulva but with a greater difference in thickness between the top and bottom of the cell in Amanita deflexa. Finally, it differs from Amanita fulva by its growth under pines. By its two-layered volva and by the change in colour of the pileus during its development, this species deviates not only from Amanita fulva but from all other European taxa of the stirps Fulva.

Additional collections examined: France, Haut-Rhin, la Gauchmatt, Soulzmatt, under Pinus nigra, 29 Oct 2019, J.-L. Muller, JMH2019039 (herb. J-M. Hanss); Vienne, Chavigny, Forêt de Mareuil, 26 Oct 2022, Y. Bellanger, JMH2022008 (herb. J-M. Hanss). Hungary, Vas County, Őrség, Orfalu, “Blueberry Trail”, 3 Oct 2022, L. Albert, B. Dima, DB-2020-10-03-1/AD52 (ELTE).


Amanita griseofulva Hanss, P-A. Moreau, Dima & D. Varga, sp. nov., Figs. 6, 7.

MycoBank: MB#846788.

Holotype: France, Mayenne, forêt d’Ombrée-d’Anjou-Combrée, under Betula pendula and Quercus petraea on oligotrophic soil with Pteridium aquilinum, A. Gasch & P-A. Moreau, 21 Aug 2021, LIP 0404490 (ex PAM21081109); GenBank PP375686 (ITS) and PP273651 (TEF1-α).

Etymology: the epithet griseofulva refers to the similarity of the species with Amanita fulva, with a distinct, greyish coloured pileus.

Diagnosis: Reminiscent of Amanita fulva by the slender habitus, but it differs by growing strictly under deciduous trees on all types of soil, by a vaginate, semi-friable to friable volva often broken into small patches, by a quickly plane-convex pileus, not fawn but grey, grey-beige, yellow-grey and sometimes also dark grey-brown to bronze-green, by a partial veil forming small white aggregates that are clearly visible on young basidiomata, by a subhymenial tree with isodiametric cells under almost all basidia and by a unique phylogenetic position.

Description: Pileus 2–8 cm in diameter, quickly plane-convex, and then slightly depressed with a small persistent umbo, grey, grey-beige, yellow-grey and sometimes also dark grey-brown to bronze-green, shortly striate at the margin in some specimens. Lamellae white, only slightly greying with age; edge with grey edge on mature specimens. Stipes 4.5–10-cm high, often quite thin but sometimes more solid which can give an aspect of Amanita fulvoides, most often white, with tiny clusters of white partial veil remains visible on young mushrooms. Dark coloured collections may have a coloured stipe with age. Volva 1.2–2-cm high, vaginate, fragile, thin, whole (type III) or fragmented (type I), white, stained with brown spots. Basidiospores 8.8–13 × 8–12 µm, globose to subglobose, Lav = 9.76 µm, Wav = 9.32 µm, Qav = 1.03. Basidia of stocky appearance because of the small length/width ratio, clearly capitated then rather abruptly tapered down, 55–70 × 14–20 µm, mostly 4-spored but 2-spored basidia are relatively frequent. Sterigmata 5–6-µm long. Lamellar trama about 100 μm broad, reduced to a mediostratum whose elements show an essentially parallel arrangement. It is composed of 4–6-µm thick hyphae, either septate with short elements to 40 µm in length or with very long elements. Many physaloid cells mostly elongated, the largest around 8 × 20 µm or more rarely spherical, 25 × 25 µm. The whole forms a solid matrix that is difficult to decompose. Subhymenium under ripe basidia isodiametric, type 4 or 5, subhymenial cell 15 × 15 µm or 20 × 20 µm, more rarely ramose and fixed on an isodiametric cell. Under the immature basidia, the subhymenium is always cellular. Lamellar edge wrapped in a gelatinous matrix from which sometimes a few subspherical marginal cells emerge, 30–20 × 25–18 µm, pedunculated, grouped in bundles or irregularly distributed on the edge (in some places of the lamella edge marginal cells lacking). Pileipellis 150–180 µm broad, forms an ixocutis with a thin gelatinous layer, comprising some thin hyphae 1–3 µm broad, covering a cutis of 3–5 µm broad hyphae, almost parallel, arranged in twist (spiral) ensuring mechanical coherence, with some very elongated acrophysalids, e.g. 80 × 10 µm. Transition zone with context non-observable. Context of pileus mostly composed of elongated physaloid cells, e.g. 140 × 30 µm, 100 × 40 µm, 45 × 17 µm and sometimes forming chains and hyphae up to 4–5-µm thick. The whole is weakly entangled. Volva ca. 600 µm broad with a high density of quite massive, largely ellipsoid, physaloid cells, e.g. 80 × 60 µm, 50 × 30 µm, more rarely almost spherical, 35 × 30 µm, maintained by 4–5-µm thick hyphae, strongly septate, with short, 50–60-µm long segments. Acrophysalides sometimes present, e.g. 30 × 9 µm, 45 × 40 µm. The entanglement is a little stronger outward.

Phylogenetic relationship: Amanita griseofulva is rather isolated, representing the most basal clade in stirps Fulva, differing by at least 16 substitution and indel positions (95% similarity) from the closest species (A. pachycolea) in the ITS region. Intraspecific genetic variability is very low, most sequences are identical, and few have 1–2 nucleotide differences.

Habitat and distribution: in mixed deciduous forests under Quercus, Carpinus and Fagus on all types of soils, from calcareous to oligotrophic soils. Presumably not unfrequent in Western and Central Europe, but verified data are scattered to date with known occurrences from France, Germany, UK (soil sample – GenBank: KM374392) and Hungary where it seems widespread from the westernmost to the easternmost areas. Fruiting in summer.

Comments: Macroscopically, A. griseofulva forms small- to medium-sized basidiomata, characterised by a lowly umbonate pileus which can display a variety of colours but most often rather pale, a rather slender stipe, most often white, covered with a slight partial veil forming white to light grey punctuations of a unique appearance among the taxa within the stirps Fulva, and a fragile, rust-stained, vaginate volva, mainly entire but also dissociated pieces on pileus or around base. The only medium entanglement of the volva does not always seem to be able to ensure mechanical strength of a tissue in which physaloid cells dominate. The subhymenium is cellular, a relatively rare property in the sect. Vaginatae and therefore remarkable. This species has many microscopic similarities to A. fulvoides. In case of doubt, the structure of the pileipellis will make it possible to distinguish them.

Additional collections examined: France, Nord, Saint-Amand-les-Eaux, Forêt domaniale de Raismes-Saint-Amand-Wallers, Drève des Prés Charniers, parcel 309, under Quercus petraea and Carpinus betulus on clay-limestone, soil stirred up by wild boars, P-A. Moreau, 18 Sept 2016, PAM16091803 (LIP0401182); Beuvry-la-Forêt, forêt domaniale de Marchiennes, parcel 15, under Quercus petraea and Betula pendula on oligotrophic soil, P-A. Moreau, 12 Sept 2019, PAM19091201 (PAM19091201). Hungary, Heves County, Mátra Mts, Parádfürdő, under Quercus cerris and Q. petraea, A. Nagy, B. Dima, 5 July 2020, DB-2020-07-05-6/AD37 (ELTE); Mátra Mts, Parádsasvár, under Fagus sylvatica and Quercus petraea, A. Nagy, B. Dima, 25 July 2020, DB-2020-07-25-2/AD09 (ELTE); Baranya County, Mecsek Mts, Pécs, Éger-tető, under Quercus cerris and Q. petraea, B. Szűcs, B. Dima, 27 Jun 2020, DB-2020-06-27-4 AD14/(ELTE); Borsod-Abaúj-Zemplén County, Rudabánya Mts, Zubogy, under Quercus petraea and Carpinus betulus, I. Fedor, 7 July 2018, DB-2018-07-07-6/AD24 (ELTE); Rudabánya Mts, Zubogy, under Quercus petraea and Carpinus betulus, M. Németh, 7 July 2018, DB-2018-07-07-3/AD15 (ELTE); Rudabánya Mts, Zubogy, under Quercus petraea and Carpinus betulus, B. Bognár, 7 July 2018, DB-2018-07-07-05/AD21 (ELTE); Vas County, Őrség, Bajánsenye, under Quercus petraea, Carpinus betulus, Fagus sylvatica and Pinus sylvestris, B. Dima, D. Varga, 29 Jun 2022, DB-2022-06-29-1 AD143/(ELTE).


Amanita griseofulva f. albida Hanss & Dima, f. nov., Fig. 6f.

MycoBank: MB#850078.

Holotype: Hungary, Borsod-Abaúj-Zemplén County, Zemplén Mts, Újhuta, under Quercus petraea, Pinus sylvestris, Carpinus betulus and Betula pendula, A. Nagy, B. Szűcs, B. Dima, 31 July 2021, DB-2021-07-31-2/AD115 (BP); GenBank PP375682 (ITS), PP375222 (LSU), PP358483 (RPB1) and PP273650 (TEF1-α).

Etymology: the epithet albida refers to the white basidiomata of this form.

Diagnosis: Differs from typical Amanita griseofulva by an entirely white pileus.

Comments: Genetically this apigmented form of A. griseofulva shows no difference from the sequences of the typical form and the two collections studied are nested within the A. griseofulva clade.

Additional collection examined: Borsod-Abaúj-Zemplén County, Zemplén Mts, Újhuta, in mixed forest, K. Gábor Békésné, 31 July 2021, DB-2021-07-31-3/AD117 (ELTE).


Amanita opaca Hanss, Dima, Bidaud & D. Varga, sp. nov., Figs. 8, 9.

MycoBank: MB#846789.

Holotype: France, Ain, Marchamp, locality “le Moyeux”, alt. 800 m, under Quercus pubescens and Carpinus betulus on calcareous soil, A. Bidaud, 12 Aug. 2011, LIP 0004500 (isotype AB11-08-60 in herb. A. Bidaud); GenBank PP375802 (ITS).

Etymology: Latin epithet opacus, understood in the sense dull, refers to the peculiar aspect of pileic surface on all studied collections.

Diagnosis: differs from the closely related Amanita coryli by a slightly more robust habitus, by a dull pileus with two or more colours, and by larger spores. The rather fragile, white, sometimes yellow-spotted volva has a particular microscopic structure, mainly composed of oval, physaloid cells wrapped in tendrils of helical hyphae, arranged in parallel to the stipe, and as long as the height of the volva.

Description: Pileus 2–10 cm in diameter, first conical to parabolic, then plane-convex, gradually becoming flat; surface smooth or with small to wide patches of universal veil, with a dull appearance, always composed of two main colours one of which being grey, intimately mixed in certain areas where they form a rich palette of gradations, darker in the centre and on the striate margin. Lamellae free, crowded, whitish. Stipes 3–12-cm high, rather thin but sometimes more solid, entirely white, smooth to finely floccose. Volva 1–3-cm high, of a particular type II, long vaginated on the bottom then opening into a short collar, fragile, thin, sometimes fragmented (type I), white and sometimes stained with rusty spots. Basidiospores 10.6–13.5 × 10–12.6 µm, globose to subglobose, Lav = 12.04 µm, Wav = 11.20 µm, Qav = 1.07. Basidia generally quite long with an elongated and slender base, 70–90 × 20–25 µm, rarely more squat, 60–70 × 20–22 µm. Lamellar trama from about 280-µm thickness at the base to 90 µm towards the tip, little differentiated where we can distinguish a thick mediostratum of about a half of the thickness, relatively parallel to the axis with few physaloid cells, oval 28 × 40 µm or 37 × 45 µm to clearly elongated (40 × 55 µm) and hyphae of 2–4(5)-µm thick. The hymenophorus has same components of the same measurements. Its physaloid cells are more and more numerous as we approach the subhymenium, with an angle of orientation varying from parallel to almost perpendicular to the axis. The subhymenial stratum appears to be cellular due to a large number of spherocysts, but the subhymenial tree is relatively short with branching elements measuring about 5 × 10 μm. Sometimes the subhymenial tree is reduced to a single cell, connected to long, parallel to the edge of the lamella, hyphae. Lamellar edge gelified or not is according to the examined samples. The 60–70-µm gelified layer does not contain hyphae or marginal cells. Below, the basidia are mixed with marginal cells of various forms, ampuliform, sometimes large (25 × 45 µm), subglobular or oval, 20–30-µm high with a short or long pedicel, and then septate, formed by several small cylindrical cells (e.g. 6 × 6 µm). Pileipellis about 200-μm thick, an ixocutis composed of a gelatinized outer matrix, up to 100-μm thick with a few floating hyphae 2–3-μm thick and a cutis 80–100-μm thick composed of hyphae 3–5-μm thick, weakly twisted in a spiral, mixed with a few elongated physaloid elements, e.g. 70 × 20 µm, 100 × 20 μm or 160 × 40 µm. In the inner part, the physaloid elements become more numerous. Absence of differentiated subpellis. Context of pileus consists mainly of acrophysalids of remarkably broad size, elongtated (e.g. 280 × 60 µm), sometimes more ovoid (e.g. 320 × 100 µm or 160 × 60 µm), sometimes forming chains of some elements, weakly connected by slender hyphae 6–12-µm wide and globally faintly resistant. Volva thick 600–700 µm, formed of a densely entangled tissue of mixed elements, apparently without orientation. A very thin section and a slight dislocation allows to observe that it contains a large number of physaloid cells generally oval of 15–45 × 20–70 µm with a Q of 1.2–1.8, more rarely spherical (for example 50 × 50 µm or 30 × 30 µm) or very long (e.g. 140 ×12 µm or 150 × 45 µm for the internal ones) wrapped by 3–7-µm-thick hyphae forming tight tendrils, mostly longitudinally oriented. Externally and internally about 100 µm broad, the tissue is even denser but without this changing its structure.

Phylogenetic relationship: Amanita opaca belongs to the stirps Coryli, differing by 7–8 nucleotide and indel positions (98% similarity) from A. cistetorum, 12 changes (97% similarity) from Amanita sp. (‘saltpointensis’) from USA, and at least 17 changes (96% changes) from A. coryli from Europe. In our multigene analysis, A. opaca forms the sister clade of A. cistetorum with strong support. Intraspecific genetic variability is low, most sequences are identical, and only in few cases, 4–5 indel differences have been detected (Fig. 3).

Habitat and distribution: Mixed thermophilous deciduous forests under Quercus spp. and Carpinus betulus but possibly also with Betula pendula. One sequence-verified record is originated under Pinus nigra as host tree (GenBank: OQ327043). Known from France, Germany, Greenland (UNITE: UDB002307), Hungary, and the UK (Andy Overall, pers. comm.), but with several records, thus it can potentially be more widespread in Europe.

Comments: Amanita opaca is one of the many rather anonymous greyish, greyish-brown ‘vaginata’ species occurring in thermophilous woodlands of Europe. Currently, morphological differentiation towards other similar species is often difficult. The closely related A. coryli has whitish volva, less robust habit, an always strictly vaginate volva and smaller basidiospores (Figs. 15, 16 and 17). The recently described A. prudens (Plaza 2022) has a first hemispherical pileus with a moiré aspect, somewhat more or less iridescent. In the beginning of the maturity phase this feature is matched with a shiny to silky character and then the pileus flattens, gradually becomes duller and then of more banal appearance. It is also belonging to stirps Coryli, can be distinguished from A. opaca based on macromorphology when young basidiomata are studied, in addition it also forms smaller basidiospores than A. opaca (Figs. 15, 16 and 17). Tight tendrils enveloping groups of physaloid cells (and forming cylinders) also exist in A. coryli and A. prudens. But it is in A. opaca that they are the tightest and most typical. For this reason, we propose to call this original volva structure “opaca type”.

Fig. 15
figure 15

Comparison of basidiospore length data. Graph showing the mean and standard deviation of spore lengths of 32 species of Amanita sect. Vaginatae. Following the ANOVA analysis, Tukey’s post hoc test was used for pairwise comparisons at p < 0.05 significance level

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Fig. 16
figure 16

Comparison of basidiospore width data. Graph showing the mean and standard deviation of spore width of 32 species of Amanita sect. Vaginatae. Following the ANOVA analysis, Tukey’s post hoc test was used for pairwise comparisons at p < 0.05 significance level

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Fig. 17
figure 17

Comparison of Q-value of spores. Graph showing the mean and standard deviation of basidiospore Q-value of 32 species of Amanita sect. Vaginatae. Following the ANOVA analysis, Tukey’s post hoc test was used for pairwise comparisons at p < 0.05 significance level

Full size image

Additional collections examined: France, Isère, Vézeronce-Curtin, les Rochettes, alt. 240 m, A. Bidaud, 3 May 2012, AB12-05-05 (LIP 0004498); Haute-Saône, Lure, water sport centre, in partly planted grove with Carpinus betulus, Betula pendula, Acer pseudoplatanus, Quercus robur, Salix sp. and Picea abies, alt. 300 m, J-M. Hanss, 12 Jun 2020, JMH2020007 (in herb. J-M. Hanss). Hungary, Veszprém County, Bakony Mts, Úrkút, Kab-hegy, under Quercus sp. and Carpinus betulus, L. Vajda, I. Sárközi, G. Mokánszki, 19 Jun 2021, DB-2021-06-19-5/AD104 (ELTE), DB-2021-06-19-1/AD100 (ELTE), DB-2021-06-19-3/AD102 (ELTE), DB-2021-06-19-2/AD101 (ELTE); Komárom-Esztergom County, Vértes Mts, Tatabánya, under Quercus sp., Gy. Vidra, 13 Jun 2021, DB-2021-06-13-4/AD108 (ELTE); Vas County, Őrség, Velemér, under Carpinus betulus, A. Nagy, B. Dima, 27 Sept 2020, DB-2020-09-27-3/AD51 (ELTE); Borsod-Abaúj-Zemplén County, Rudabánya Mts, Zubogy, under Quercus petraea and Carpinus betulus, I. Fedor, 7 July 2018, DB-2018-07-07-7/AD23 (ELTE); Pest County, Buda Mts, Budakeszi, Hidegvölgy, under Quercus cerris and Q. petraea, A. Nagy, B. Dima, 28 Jun 2020, DB-2020-06-28-8/AD86 (ELTE); Heves County, Mátra Mts, Szuha, under Betula pendula, Populus tremula and Carpinus betulus, A. Nagy, B. Dima, 2 Aug 2020, DB-2020-08-02-3/AD39 (ELTE).


Amanita opaca f. cettoi Hanss & Dima, f. nov., Fig. 8h–i.

MycoBank: MB#846790.

Holotype: France, Doubs, Laviron, in a mixed forest on calcareous soil, S. Rousset, 5 Oct 2016, LIP 0004499 (isotype JMH2016027 in herb. J-M. Hanss); GenBank MN493559 (ITS, as ‘A. supravolvata’).

Etymology: attributed to the Italian mycologist Bruno Cetto, who illustrated this species under the erroneous name “Amanitopsis alba Gillet” in volume 3 of the series “I funghi dal Vero”.

Diagnosis: differs from Amanita opaca by a uniform white colour of the basidiomata, the volva may be stained yellow like that of the main form.

Comments: Phylogenetically this apigmented form of A. opaca shows no difference from the sequences of the main form and are nested within the clade of A. opaca.

Discussion

Our study provides a new framework for the phylogenetic relationships of Amanita sect. Vaginatae in Europe, especially focusing on Central European data. The original aim of the study was to revise the Vaginatae species in Hungary; however, the project had soon become international including specimens and sequence data from several other countries (i.e. Austria, France, Italy, Norway and Romania). Based on multigene analyses of nrDNA ITS, nrDNA LSU, RPB1 and TEF1-α sequences, we provide new phylogenetic data of 36 European species from 151 studied material. We, furthermore, recognized six larger clades including 22 stirps (12 of them are newly identified), which help us to classify species into more natural groups within the section. Comparing to Hanss and Moreau (2020) who recognized five clades and 11 stirps, all of those were recovered in our analyses too, and we followed their classification. Our 36 species were clustered into the stirps Albogrisescens, Arctica, Argentea, Betulae, Ceciliae, Coryli, Crocea, Friabilis, Fulva, Lividopallescens, Magnivolvata, Mairei, Nivalis, Simulans, Spadicea, Submembranacea and Vladimirii. In stirps Fulva and Coryli, we describe three new species to science (A. deflexa, A. griseofulva and A. opaca) and apigmented forms of the two last (A. griseofulva f. albida and A. opaca f. cettoi). All new species seem to be widespread in Europe but mostly overlooked due to their similarity with related species. Amanita deflexa has likely been often confused with A. fulva from which it mainly differs by ecology, colour of inner surface of volva, microscopy of volva and ITS sequence. The two other species (A. griseofulva and A. opaca) described here, due to their greyish pileus colours might have so far been overlooked and were hidden under the collective name Amanita vaginata s. lat. The revision and typification of this name will be the object of a separate project.

Additions to microscopical observations

The microscopy applied in this study is the one taught by C. Bas and adopted by current amanitologists. Thin sections reveal an astonishing variability of the histological aspects of certain tissues which, combined with the observation of the spores, of the subhymenium, of the edge of the lamellae, as well as of macroscopic particularities make possible the conception of identification keys to the European Vaginatae. Such a key, focused on silvery-capped species, may be found in Hanss and Moreau (2020).

Most often, detailed microscopy can alone bring light to species identification in Amanita sect. Vaginatae, but doing this properly, requires a good knowledge of the morphology of Amanita, precise work as well as precise cutting equipment. Experience is acquired fairly quickly through practice. During the study of very numerous species, we observed that the angle of divergence of the physaloid elements of the lamellar trama becomes smaller to zero during drying. At rehydration, it does not return to its initial value and may, in some cases, remain at zero. The vacuolar pigments of the rehydrated hyphae in Amanita are hyaline. When grouped into large packets, they may have a yellowish shade. A brown intracellular necropigment may be present in some sphaerocysts of volva (e.g. in species of Clade 4). The volva of Amanita is an organ with highly variable microscopic structure. The volva is an important part of the universal veil protecting during the early development of the fungus. During the growth of the amanitas of the section Vaginatae, the ring does not develop, and the partial veil is distributed on the surface of the stipe and on the inner surface of the lower volva. This partial veil is a foreign element to the universal veil and because of that it disrupts the understanding of the structure of the universal veil. Because of this reason, we study the structure of the volva in its upper part. At the bottom of the volva, there is the limbus internus whose study can sometimes be of interest. Our method is to obtain the following information by a fine cut (and not crushing): the composition of the volva from whole elements, the arrangement of the elements in relation to each other, their orientation and their degree of entanglement. This is infinitely richer in information than those with crushing which gives a slurry in which we no longer recognize the structure.

Basidiospore size and shape as diagnostic character in Amanita sect. Vaginatae

Albeit macromorphologically distinct and phylogenetically unrelated, the three new species described in this study display very similar basidiospore shape (globose to subglobose, with a mean Q value between 1.03 and 1.07), and sizes (ranges from 7–13.5 × 7–12.5 µm). Nevertheless, there are some differences of the features: the spore length and width of A. deflexa is significantly different than those of other species in stirps Fulva, except A. umbrinolutea, while in Q value, there is no difference towards the closely related species in this stirps. Amanita griseofulva also differs in its spore length and width from all species in the stirps Fulva, except A. fulva, but we found no difference in Q-value from other species in this stirps. In the case of A. opaca, when comparing basidiospores within the stirps Coryli which the species belongs to, we found significant differences in spore length and width, but no differences in Q values of A. opaca, A. coryli and A. prudens. However, with its greyish brown pileus colour, A. opaca can be very similar to other, more distantly related Vaginatae species, where basidiospores are also very similar (Figs. 15, 16 and 17). Albeit overlapping, there were significant differences of the basidiospore length, width and Q value of 32 Vaginatae species studied here revealed by the statistical analyses (Table S1). There are differences among all those values analysed; nevertheless, when significant pairwise differences are considered, there is no species which differs from all or similar to all other species studied here (Figs. 15, 16 and 17). The significant pairwise differences and similarities of species studied do not correspond with the main clades/stirps.

In conclusion, based on our statistical analysis, we found that basidiospore length and width data as well as Q values as diagnostic characters in the identification of the studied species cannot be considered as unambiguous overall taxonomic feature in Amanita sect. Vaginatae, and this observation is in line with the previous study of A. lividopallescens complex (Vizzini et al. 2016), where the authors stated that the spore-shape and presence/absence of lamellar sterile elements as specific distinguishing characters within section Vaginatae was overemphasized in the past (e.g. Romagnesi 1992; Tulloss 1994; Contu 2000, 2003; Neville and Poumarat 2009).

Amanita sect. Vaginatae in Hungary and the benefit of national data to improve diversity knowledge

Despite their taxonomically challenging nature and their widespread occurrence in Hungary, little local data of species within the section Vaginatae were available, and no study on their taxonomy and phylogenetics was carried out before our work. These species are found in many habitats throughout the country, especially in hilly and mountainous forests dominated by tree species belong to the genera Fagus, Quercus, Carpinus, Betula, Pinus and Picea. Based on morphological identification, 15 taxa were documented from Hungary: Amanita argentea, A. battarrae, A. umbrinolutea, A. beckeri, A. ceciliae, A. crocea, A. dryophila, A. friabilis, A. fulva, A. lividopallescens, A. lividopallescens var. tigrina, A. mairei, A. pachyvolvata, A. vaginata and A. vaginata f. alba) (Babos 1989; Rimóczi 1994; Pál-Fám 2002; Pál-Fám and Lukács 2002; Benedek and Pál-Fám 2006; Vasas and Locsmándi 2009; Lukács 2010; Albert 2011; Dima et al. 2013; Folcz et al. 2013), but so far these identifications have not been verified by molecular taxonomic methods.

Our comprehensive study revealed that Hungary hosts at least 25 species of Amanita section Vaginatae of which only eight taxa, A. ceciliae (partly as A. inaurata), A. crocea, A. dryophila, A. friabilis, A. fulva, A. lividopallescens, A. battarrae (as A. pachyvolvata) and A. umbrinolutea have previously been reported based on morphological species concept. As a result, we documented 17 species new to Hungary: Amanita albogrisescens, A. alseides, A. calida, A. coryli, A. deflexa, A. fulvoides, A. griseofulva, A. herculis, A. huijsmanii, A. opaca, A. prudens, A. simulans, A. spadicea, A. suberis, A. sublividopallescens, A. subnudipes and A. supravolvata.

The presence of A. argentea, A. beckeri and A. mairei based on the currently revised species concepts has not yet been confirmed from Hungary. The names A. argentea and A. mairei in earlier Hungarian publications most likely harbour a number of greyish Vaginatae species, but based on DNA sequence data, none of the two species occurs in Hungary to date. The hardwood-associated A. argentea and A. huijsmanii have commonly been confused until the epitypification of A. argentea in Hanss and Moreau (2020). Similarly, the Pinus-associated A. mairei and A. supravolvata have often been misinterpreted until the study of Hanss and Moreau (2020). In the literature, we can find two other cases of confusion: (i) between silvery species and grey species that are never silvery (e.g. A. argentea and A. coryli) and (ii) between silvery species and coloured species that are occasionally silvery (e.g. A. argentea and A. albogrisescens). Our study also confirmed that all Hungarian records of A. beckeri (Babos 1989) represent A. fulvoides. A fleshy species with orange pileus and whitish stipe is often recorded on calcareous soils under Quercus in Hungary under the name Amanita crocea, but originally this species is associated mainly with Picea or Betula in acidophilous habitats and has usually orange hues on stipe. Based on our sequence data, all Hungarian records identified as A. crocea from Quercus forests likely represents A. subnudipes (with a whitish stipe), a species described from deciduous woodlands from France (Romagnesi 1982, Tulloss 2000). The occurrence of A. crocea s. str. was confirmed based on one collection from the northern mountains under Betula and Picea (Figs. 3, 12b).

The wide morphological concept of Amanita vaginata is currently not fixed. This classical binomial appears in several distantly related lineages within sect. Vaginatae across the phylogenetic tree (Fig. 3). Our own records of white forms of Vaginatae species (often referred to A. vaginata f. alba or A. alba) represented multiple species belonging to A. huijsmanii, A. griseofulva, A. opaca, A. coryli, A. magnivolvata and A. simulans.

All previous records of A. battarrae turned out to be A. umbrinolutea, while the specimens of A. pachyvolvata represented more species including A. battarrae (syn.: A. magnivolvata), A. spadicea and A. supravolvata. Based on the recent epitypification of Amanitopsis vaginata var. battarrae and Amanitopsis pachyvolvata by Hanss and Moreau (2022), and the type sequence of A. magnivolvata in Hanss and Moreau (2020), the three names are here established as synonyms. Amanita battarrae is associated with deciduous trees, while the other taxa are associated with conifers. Because Riel (1907) raised Amanitopsis battarrae to species rank, the legitimate name to be applied to this taxon is Amanita battarrae (Boud.) Bon. Finally, Amanita lividopallescens var. tigrina were synonymized with A. lividopallescens in Vizzini et al. (2016), we, however, recognized two distinct lineages where specimens with ‘lividopallescens’ morphology clustered (Fig. 3). Amanita lividopallescens was epitypified in Vizzini et al. (2016) with an ITS sequence. From the 13 Hungarian collections studied here, only three clustered in the clade where the epitype sequence occurred, and ten sequences clustered to another, sister clade which was recently described as A. sublividopallescens (Fig. 3) by Migliozzi and Di Palma (2024). National records like in this study can help to improve larger-scale taxonomic and biodiversity knowledge when the data are included to an international collaboration.