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Article

Taxonomy, Phylogeny and Ecological Assessment of the Truffle Genus Genea in Central Europe with a New Species and a New Record

by
Swagata Chakraborty
1,2,*,
Shruti Anand Tirpude
2,
Balázs Domonkos Péter
2,
Getnet Chekole Walle
2,3,
Akale Assamere Habtemariam
4,
Alfonz Kedves
5,6,
Máté Balogh
6,
Zoltán Kónya
6,7 and
Zoltán Bratek
1,2
1
Doctoral School of Biology, University of Szeged, Dugonics Square 13, H-6720 Szeged, Hungary
2
Department of Plant Physiology and Molecular Plant Biology, Eötvös Loránd University, Pázmány Péter Sétány 1/C, H-1117 Budapest, Hungary
3
Department of Biology, University of Gondar, Gondar P.O. Box 196, Ethiopia
4
Department of Biology, Mekdela Amba University, Gimba P.O. Box 32, Ethiopia
5
Institute of Animal Science and Wildlife Management, Faculty of Agriculture, University of Szeged, Andrássy út 15, H-6800 Hódmezővásárhely, Hungary
6
Department of Applied and Environmental Chemistry, University of Szeged, Rerrich Béla Tér 1, H-6720 Szeged, Hungary
7
HUN-REN-SZTE Reaction Kinetics and Surface Chemistry Research Group, Rerrich Béla Tér 1, H-6720 Szeged, Hungary
*
Author to whom correspondence should be addressed.
Diversity 2026, 18(6), 360; https://doi.org/10.3390/d18060360 (registering DOI)
Submission received: 30 April 2026 / Revised: 5 June 2026 / Accepted: 9 June 2026 / Published: 12 June 2026
(This article belongs to the Section Microbial Diversity and Culture Collections)
Browse Figures
Versions Notes

Abstract

Hypogeous ascomycetous fungi (truffles) are challenging to study because they produce underground sporocarps that may not be encountered during traditional fungal surveys. Genea is one such genus that has garnered considerable attention over the past decades due to its role as an ectomycorrhizal partner and contribution to nutrient cycling and ecosystem stability. Yet, very limited information is available about its taxonomy, phylogeny and ecology worldwide. The current study aims to expand the known distribution of Genea species in Central Europe by integrating morphological, molecular and ecological analyses of new collections as well as the assessment of herbarium materials. Light microscopy and SEM were used to determine morphological characteristics along with FT-IR (Fourier transform infrared) spectroscopy measurements, which proved to be a powerful tool for species differentiation. Molecular phylogenetic analyses were conducted using the internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) and D1/D2 domain of the large subunit (28S) of nuclear ribosomal DNA sequences to confirm species identity. In this study, a new species, Genea szemereiensis, along with the first report of Genea pinicola from Hungary, was made. In addition, the ecological parameters of the species, including habitat, altitude, soil nutrients and pH, were revised, which has not been reported previously in detail for this genus.

1. Introduction

The genus Genea Vittad, dedicated to zoologist Dr. Joseph Gené, comprises a group of hypogeous fungi within the division Ascomycota [1] with G. verrucosa Vittad as the type species. This genus, characterized by hypogeous ascomata, often folded and with a tuft of basal hyphae, is commonly found in the Mediterranean terrain. The peridium is typically black, brown, or reddish brown and is covered with warts and an apical opening [2]. It consists of an organized hymenium covered by an epithecium formed by paraphyses at the peridial junction [1] with the asci usually containing eight ornamented ascospores, which are inamyloid [3]. The genus Genea is often understudied and less explored when compared to the truffles, which are widely recognized for their economic and nutritional benefits. It has been recorded from Europe, North America, and recently from parts of Turkey and especially from Mediterranean and temperate forest zones. The distribution of Genea in mainland Asia, however, remains poorly documented.
Over time, the taxonomic classification of Genea has undergone several modifications to gain proper insight into its phylogenetic relationships. Trappe originally placed Genea in the family Geneaceae [4]; however, due to some morphological similarity between hypogeous Genea verrucosa and epigeous Jafneadelphus echinatus Gamundi and J. argentinus Rifai, Pfister assigned Genea to Pyronemataceae based on both molecular and morphological evidence [5,6,7]. One of its most important features is that it seems particularly well adapted to a broad range of environments, as Genea forms ectomycorrhizal (ECM) symbionts with woody plants and exhibits broad ecological associations with a diverse array of host trees, including fir, larch, pine, oak, beech, birch, chestnut, hazel, hemlock, hornbeam, linden, and Douglas fir [3,8,9,10,11,12,13,14,15,16,17,18]. They are sometimes found to be abundant colonizers of EM roots in seasonally dry, high-altitude forests [19,20,21] and several forest types after wildfire [22,23,24]. Unlike many ascomycetes that rely on active spore ejection, Genea has evolved a passive dispersal mechanism. Upon reaching maturity, their ascomata produce characteristic volatile aromatic compounds that attract mycophagous animals [25,26]. Once consumed, the ascospores survive the animal’s digestive tract and are then deposited at new sites through feces, ensuring effective spore dispersal [6].
Spanning nearly two centuries, chronologically, at present, the genus includes 49 formally recognized species, described through contributions [1,3,4,6,8,9,10,11,12,13,14,15,17,18,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41].
The current work presents our study of over three hundred specimens from our herbarium collections, primarily from Central Europe, with most species being blackish and featuring strikingly different ornamentation. Previously reported species from Hungary include Genea fragrans [30], Genea lespiaultii-sphaerica, Genea verrucosa, Genea pseudoverruoca, Genea pseudobalsleyi, Genea compressa [3] and Genea cf. subbaetica. Here, we reported a new species and a new record of Genea from Hungary. The main aim of the work was to (1) perform a taxonomic revision of the genus Genea with materials collected from the Carpatho-Pannonian region and Austria, (2) elucidate the morphological parameters for distinguishing closely related species and provide an identification key, (3) sort and classify closely related species based on their chemical composition through preliminary FT-IR analysis, (4) validate the taxonomic limits and species delimitation through phylogeny and (5) clarify the vegetation types of Genea species spanning the Carpathian Basin based on their physiochemical properties.

2. Materials and Methods

2.1. Sampling

The Genea specimens (n = 319) were collected from various forest habitats in Hungary, including oak–hornbeam forests and pine-dominated areas, along with specimens from Austria, Romania, Serbia and Slovakia. All specimens were collected from natural habitats in the Carpatho-Pannonian region, primarily by certified members of the First Hungarian Truffle Society (EMSzE), using trained truffle dogs and eventually deposited in the mycotheca (ZB) of the society [42]. The fruitbodies were preserved and studied either fresh or dried. Additionally, a Microsoft Access database [43] is maintained in our lab for hypogeous fungal materials, containing comprehensive information on underground fungi. Apart from the basic data of the fruiting bodies, the database also stores records of ecological parameters, including collection dates, precise geographical coordinates, habitat descriptions, and unique identification codes as well. Soil samples were collected from the vicinity of the fruit bodies (truffle bed) from the soil layer where sporocarps were found with the help of hoes. After drying and sieving, the soil samples were packed in plastic bags and sent to the Nemzeti Élelmiszerlánc biztonsági Hivatal Élelmiszerlánc-biztonsági Laboratórium Igazgatóság Növény- és Talajvédelmi Nemzeti Referencia Laboratórium, Velence, Hungary, for analysis.

2.2. Morphological Measurement

The morphological analysis of fresh or dried ascomata from the Genea samples (n = 319) was performed using a Nikon research microscope (Optiphot 2, Nikon Co., Tokyo, Japan). Macromorphological characteristics, including the shape and size of the ascomata as well as the surface texture and coloration of the peridium and gleba, were examined and described using the Colour Identification Chart of the Royal Botanic Garden Edinburgh [44]. The sample preparations were further mounted on polyvinyl alcohol–lactic acid–glycerol (PVLG) [45] medium for measuring micromorphological characteristics like the structure and size of the peridium and the size of the asci and ascospores, along with the height and type of ornamentation. In addition, the Piximétre Version 6.1 R 560 program was used to measure spore sizes. Ascospore morphometric data, including spore length, width and ornamentation along with Q values, were recorded from 30 ascospores per species (n = 30 for all six most frequently occurring taxa; total n = 180). All measurements are presented as mean ± standard deviation (SD) with minimum–maximum ranges. (Supplementary File S1, Table S3).
Statistical differences in ascospore dimensions among the closely related species were assessed using the Brown–Forsythe test, followed by Welch’s ANOVA, given the unequal variances detected among groups. Pairwise comparisons were subsequently conducted using Dunnett’s T3 post hoc test, and the statistical significance was set at p < 0.05. All analyses were performed using GraphPad Prism version 10.0 (GraphPad Software, Boston, MA, USA).
To assess the surface features of ascospores, scanning electron microscopy (SEM) was performed using a Thermo Fisher Scientific Apreo C device operated at 10 kV. Before imaging, the dried samples were coated with a thin layer of gold using a Quorum SC7620 (Apreo C, Thermo Fisher Scientific Ltd., Waltham, MA, USA, Quorum Technologies, Newhaven) sputter coater to enhance surface conductivity.
Furthermore, structural characteristics of the fungal samples were examined using Fourier-transform infrared (FT-IR) spectroscopy. The measurements were conducted with a Bruker Vertex 70 instrument (Bruker Optics GmbH & Co. KG, Ettlingen, Germany) at a resolution of 4 cm−1, averaging 16 scans per second, within the spectral window of 2000–500 cm−1. The samples were prepared in the form of KBr pellets before analysis. The experiment was performed in triplicate for the newly described species. For G. fragrans, four representative specimens were selected amongst all other closely related taxa to account for the two previously recognized clades reported in earlier phylogenetic studies [12], ensuring adequate representation of intraspecific chemical diversity across its known lineages.

2.3. Molecular Study

Total genomic DNA was isolated once per specimen from fresh or dried ascomata with the DNeasy Plant Mini Kit (Qiagen, Courtaboeuf, France), according to the manufacturer’s instructions. Some modifications were done on the protocol, such as the addition of buffer AP1 and freezing the samples three times using liquid nitrogen before heating at 65 °C with a BIOER Mixing Block MB-102. The subsequent volumes of elution buffer (AE) were 30 μL and 50 μL. The amplified loci were the internal transcribed spacer (ITS) and the large subunit of nuclear ribosomal DNA (28S) locus. For the ITS, we used ITS1F/ITS4 primers [46] and NL1/NL4 primers to amplify the 28S region [47]. PCR was conducted under the following conditions: initial denaturation at 94 °C for 5 min; 30 cycles of denaturation at 94 °C for 30s; annealing at 55 °C for 20s; elongation at 72 °C for 50s; and, after the last cycle, a final synthesis stage at 72 °C for 1 min. For gel electrophoresis of the amplified products, 1% agarose gel was used. Staining was performed with ethidium bromide. For the purification of the PCR products, we used the QIAquick® PCR Purification Kit (Qiagen, Courtaboeuf, France). Sanger sequencing was carried out by BIOMI Ltd. (Gödöllő, Hungary).

2.4. Phylogenetic Analysis

The newly generated sequences for this study were assembled and compared with other sequences through BLASTn searches [48] and were eventually deposited in GenBank. A meticulous screening process based on similarity scores obtained through BLASTn searches from NCBI was adapted to select closely related taxa as the ingroup and the distantly related sequences as the outgroup. Each gene was compared separately for incongruence (Supplementary File S1, Figure S3). As there was no strongly supported contradiction, the datasets were considered congruent and were combined in a two-partition matrix, with missing data coded as gaps (-) in the combined alignment. Two phylogenetic trees were generated with NGPhylogeny.fr [49] based on 8 newly generated sequences for this study and 97 sequences from GenBank and UNITE [50] (Supplementary File S1, Table S1). Maximum likelihood (ML) and Bayesian inference (BI) methods were conducted to validate the existing species as well as to confirm our new species statistically. The sequences were manually checked with FinchTV 1.4.0 and aligned with MAFFT 7.407 [51] for the larger dataset with the entire genus and T-Coffee alignment [52] for the smaller tree comparing the new species with the Genea balsleyi-pseudobalsleyi complex. The aligned DNA sequences were manually corrected and edited using MEGA 6.06 [53] in case of ambiguous sites. The best-fitting substitution model for phylogenetic tree construction was chosen using jModelTest 2.1.10 [54,55]. For estimating phylogeny using maximum likelihood for the entire genus Genea, PhyML 3.1/3.0 aLRT [56] with bootstrap analysis including the Tamura-Nei (Equal Frequencies) model with invariant sites and Gamma-distributed rate heterogeneity (TN93 + I + G) [57] (1000 replicates) was implemented. In contrast, for the second tree with closely related taxa, Bayesian inference with MrBayes 3.2.6 [58] was implemented, with the K2P + G model being the optimal one according to the Akaike information criterion (AIC). The same approach was also applied to perform Bayesian inference analysis of the Genea sequences for the larger consensus dataset (Supplementary File S1, Figure S2). The likelihood of the final tree was optimized, annotated and visualized using iTOL v6 [59]. A 70% majority-rule tree was constructed, and posterior probabilities were computed. Furthermore, to establish species-level boundaries and to validate our new species within the morphologically cryptic Genea genus, species delimitation analyses were performed using the multi-rate PTP (mPTP) (http://mptp.h-its.org, accessed on 25 May 2026), which manages datasets encompassing species with varied levels of molecular diversity, and the single Poisson Tree Processes model (PTP) (http://species.h-its.org, accessed on 25 May 2026) with the Newick format tree produced by NGPhylogeny.fr.

2.5. Ecological Studies

Soil physicochemical variables were standardized to zero mean and unit variance prior to PCA and PERMANOVA to account for differences in measurement units. PCA was performed using R 4.3.1 packages (FactoMineR version(v).2.11, factoextra v.1.0.7, ggplot2 v.4.0.2, ggrepel v.0.9.6, scales v.1.4.0, dplyr v 1.1.4, etc.) [60,61] to visualize edaphic gradients among Genea species (n = 67 herbarial data, n = 146 Pluto F platform data), with 68% data ellipses indicating core dispersion of species groups. Differences in multivariate soil composition among species were tested using PERMANOVA with adonis2 (in vegan R package v.2.7.2) based on Euclidean distances and 999 permutations. Homogeneity of multivariate dispersion was assessed using betadisper (in vegan R package v.2.7.2). Pairwise PERMANOVA with adonis2 and Benjamini–Hochberg correction was used to identify species pairs with significantly different or overlapping soil physicochemical conditions. Descriptive statistics were used to visualize the distinct plant association types for the identified Genea species in Hungary (n = 61), using SPSS Version 20 and Microsoft Excel 2010.

3. Results

3.1. Morphology

Fresh or dried ascomata of over three hundred Genea species were morphologically identified as G. fragrans, G. verrucosa, G. pseudobalsleyi, G. lespiaulti-G. sphaerica, G. pseudoverrucosa, G. compressa and G cf. subbaetica (Supplementary File S1, Table S2). In the process, we introduce G. szemereiensis as a new species and G. pinicola as a new record to the fungal flora of Hungary. Genea fragrans, the most abundant species according to our herbarial data, in general, displays hypogeous, hollow, subglobose and lobed ascomata, with a peridium surface displaying a brownish-black hue and covered by tiny, blunt, inconspicuous dark warts, with a small tuft of hyphae at the base. Ascospores typically range from 32 to 39 × 21 to 27 μm [12,62] and are generally verrucose, with irregular sculptural ornamentation of unequal dimensions. (Supplementary File S1, Figure S1). Genea verrucosa represents mainly conical, irregular or pointed warts with a blackish papillate peridium and spores typically less than 32 μm in diameter [63], while G. pseudoverrucosa differs from G. verrucosa in terms of its crowded, uniformly truncated ornamentation (Supplementary File S1, Figure S1). SEM depicted G. pseudobalsleyi to have distinct polygonal, irregular and conical warts with a verrucose and warty black peridium, whereas G. szemereiensis demonstrates mainly conical and irregular ornaments along with occasionally truncated elements with statistically significant differences in spore length when compared with G. pseudobalsleyi. Welch’s ANOVA followed by Dunnett’s T3 multiple comparisons test was selected to compare the significant differences in ascospore length and width for the new species and its closest relative. Genea szemereiensis differed significantly in ascospore length from both G. pseudoverrucosa (p = 0.0211) and G. pseudobalsleyi (p = 0.0009), supporting its recognition as a morphologically distinct taxon. However, no significant differences in ascospore width were detected among G. szemereiensis, G. pseudoverrucosa and G. pseudobalsleyi (p > 0.05 in all comparisons). Genea fragrans, on the other hand, consistently displayed wider ascospore length and width compared to the other three studied taxa (p < 0.0001 in all comparisons) (Figure 1). Interestingly, another group within the G. fragrans clade was observed to be densely covered, in contrast to its original scattered ornamentation. Genea sphaerica and G. lespiaultii have identical hemispherical warts and are regularly lobed, with the higher ornamentation size of G. sphaerica being the most important differentiating factor between them. Usually, the asci in Genea depict eight uniseriate spores; however, we observed that most species displayed 4–6-spored asci, which were also included in the morphological examination. A key for identifying European Genea species based on their morphology is also presented. To further strengthen the morphological characterization, a preliminary FT-IR spectroscopic analysis was performed, revealing peak intensities at 2925 cm−1 in most species, indicating CH2/CH3 asymmetric lipid stretching [64]. Chemical differences among the studied species, particularly in the fingerprint region (1800–900 cm−1), indicated that protein-related features were more dominant in G. pseudoverrucosa, G. fragrans, and G. pseudobalsleyi compared to G. szemereiensis and G. pinicola [65]. Differences in Amide II bands, carbohydrate assignments, and CH ring vibrations (750–772 cm−1) further distinguished the new species from its closely related taxa. (Supplementary File S1, Figure S6).

3.2. Molecular Phylogeny

A total of 33 taxa from the ingroup of Genea were considered for phylogenetic analysis, with Humaria hemisphaerica (MG871304) and Humaria cazaresii (OSC 111670) serving as the outgroup to ensure proper tree rooting as the closest relatives outside the ingroup. The resulting topology demonstrated strong statistical robustness across major clades, with most deep nodes supported (BS: >70%) and internal nodes reaching maximal support (BS: 100%). The phylogeny resolves principal, well-supported higher-level lineages within Genea, each separated by long internal branches and consistently high nodal support. These lineages correspond to morphologically and ecologically coherent assemblages and reflect substantial evolutionary divergence, thereby justifying their recognition as distinct higher-level phylogenetic entities within the genus.
Accession PX756463 (ZB-6097) is unequivocally nested within the G. pinicola clade. Its placement is supported by maximal statistical values at both terminal and backbone nodes (BS: 100%) and by the sequence clustering tightly with authenticated reference sequences, without evidence of long-branch attraction, topological instability, or alternative placements in exploratory analyses. Previously found in Spain and Greece, G. pinicola here is confirmed from Hungary based on strong molecular evidence. The Hungarian accession shows no significant phylogenetic divergence from the other European representative, indicating clear conspecificity rather than a distinct sister lineage. The genetic cohesion of the clade, minimal internal sequence divergence, and uniformly strong statistical support collectively confirm that PX756463 is G. pinicola. This finding represents the first confirmed Hungarian record of the species and substantially expands its known biogeographical range. The absence of phylogeographic structuring within the clade suggests either recent long-distance dispersal—potentially mediated by animal vectors or host plant associations—or a previously unrecognized Holarctic distribution obscured by limited sampling.
A second major phylogenetic novelty is the recognition of G. szemereiensis sp. nov. (herbarium code ZB-6043), which forms a strongly supported, independent lineage that is sister to G. pseudobalsleyi. The node separating ZB-6043 from G. pseudobalsleyi receives high statistical support (BS: 89%; PP: 0.985), and the branch connecting them is comparatively long, indicating substantial genetic divergence. The lineage corresponding to accession PX756462 (ZB-6043) is monophyletic, genetically cohesive, and topologically stable across analyses, with no sequences from other regions clustering within it. The absence of intraspecific ITS variation supports the recognition of our species. The combined evidence of reciprocal monophyly, strong nodal support, and clear branch length differentiation fulfills contemporary phylogenetic species recognition criteria, thereby justifying its formal treatment as a new species-level entity. The resulting topologies from ML and BI analyses are shown with BS above 70% and BPP greater than 0.50 (Figure 2 and Figure 3). Species delimitation analyses using mPTP and bPTP split the Genea szemereiensis cluster as a distinct unit relative to Genea pseudobalsleyi, even though the short branch lengths suggest relatively recent divergence. The phylogenetic tree placed Genea szemereiensis isolates PX756462, PZ341863, KJ938867 and KJ938868 within a well-supported monophyletic clade, clearly separated from the closely related G. pseudobalsleyi clade, thereby identifying this cluster as an independent operational taxonomic unit, consistent with species-level differentiation. (Supplementary File S1, Figures S4 and S5).

4. Taxonomy

Genea szemereiensis S. Chakraborty & Z. Bratek, sp. nov (Figure 4)
Mycobank MB862755
Etymology: Named after mycologist Szemere László, who pioneered the study of the hypogeous fungi in the Carpathian basin.
Diagnosis: Subglobose to irregularly lobed hypogeous ascomata with a black, scale-like, warted, single-layered peridium, morphologically similar to G. pseudobalsleyi, where the ascomata similarly line the peridium wall projections within the inner chamber. Ascospores measure less than 30 μm in diam. and are densely covered by mainly conical and irregular ornaments along with occasionally truncated elements about 2.8–3 μm high.
Type: Somogybabod, Somogy county, Hungary, 46°40′10.7″ N, 17°46′36.566″ E, 23 October 2023, leg. M. Horváth, collected from sandy soil, Holotype ZB6043, GenBank accession numbers PX756462 (ITS), PZ341863 (LSU).
Description:
Subglobose, hypogeous, black ascomata and moderately lobed, up to 6–7 mm in diam., covered homogeneously with small polygonal black warts that measure around 0.4 × 0.3 mm, along with a small basal tuft of hyphae. Single-layered pseudoparenchymatous peridium around 150 μm thick, usually composed of darker angular cells in the outer part and subglobose hyaline cells in the inner part measuring 22–35 μm. Multiple chambered inner cavity that forms projections, giving it a somewhat brain-like appearance. Gleba is pale yellow to yellowish white. The inner chamber also displayed small, irregular warts similar to those on the outer peridium surface. Blackish, pseudoparenchymatous epithecium is around 50–60 μm thick with angular cells that measure 19–27 μm in diameter. Asci are cylindrical and irregular, 237–225 × 25–36 μm, containing mostly eight uniseriate spores along with occasional four- or six-spored asci. Paraphyses cylindrical, occasionally septate, 30–55 × 2–4.1 μm. Ascospores measure (23–)24.6–26.9(–28) × (16–)17.6–21.4(–22) μm, Qav = 1.32 (n = 30), subglobose to ellipsoidal, hyaline or pale yellowish in color, ornamented by conical and irregular warts, occasionally truncated, measuring 2.8–3 μm high, and 2–2.5 μm wide. Odor remains pleasant to somewhat unremarkable.
Ecology: Mainly in Carpino-Quercetum, from late summer to early winter (August-December). Apparently, forming ECM symbiosis with Quercus species.
Distribution: All of the examined and formally described material originated from Hungary. However, GenBank and the UNITE database suggested that the species were also found in France (OM749740 as G. pseudobalsleyi) and in Romania (undescribed, UBD0786859, Quercus root tip).
Notes:
This species is morphologically similar to G. pseudobalsleyi (Agnello, Bratek & J. Cabero) because of its black peridium surface, spore size, and spore ornamentation. It shows similarity with G. pseudoverrucosa in terms of its densely covered ornamentation and to G. verrucosa in terms of its conical and irregular ornamentation. However, G. szemereiensis, with densely covered ornamentation, differs from G. verrucosa because of its scattered ornamentation. Macroscopically, it is likely to be G. pseudoverrucosa but differs in spore ornamentation, as G. pseudoverrucosa has uniformly truncated and irregular spores, whereas G. szemereiensis shows conical and irregular ornamentation with occasionally truncated elements. Unlike G. balsleyi, with a two-layered peridium, G. szemereiensis demonstrates a single-layered peridium.
Additional materials examined—HUNGARY: 1-Baranya County, Ormánság, Marócsa, hypogeous under Quercus robur, 20th November 2007. (ZB-3804), Genbank accession number KJ938868 (ITS); 2-Baranya County, Marócsa, hypogeous under Quercus robur, 29 October 1998 (ZB-1458), Genbank accession number KJ938867 (ITS)+.
Genea pinicola V. Kaounas, J. Cabero & F. García 2014 (Figure 5)
Original description: Hypogeous brownish ascomata with a single inner chamber lacking peridium wall projections; ascospore lengths measure less than 32 μm and are ornamented with cylindrical warts 1–3 μm. It is apparently associated with Pinus sp., with or without other tree species.
Notes: The ascomata of our reported specimen are hypogeous and subglobose to depressed, measuring 7 mm in diam. and yellowish brown, brown or reddish brown in color when young; however, they are black when mature. The peridium is finely warty and rough with a large apical opening, as well as a basal tuft of hyphae. The peridium is two-layered, 208–280 μm thick, formed by an external pseudoparenchymatic layer, 120–180 μm thick, composed of hyaline subglobose or angular cells, and generally measures 22–40 × 25–32 μm; the internal layer is a prosenchyma, 70–90 μm thick, with bulging scattered cells. The single inner chamber lacks conspicuous wall projections, is warted, and is similar in color to the peridium. The hymenium remains arranged in a palisade, with interspersed septate, filiform paraphyses measuring 255–400 × 1–3.6 μm, and forming a pseudoparenchymatous epithecium above the asci. Asci cylindrical, 223–342 × 24 μm, containing eight uniseriate spores. Ascospores ellipsoid, 25.7–28.9 × (15.0–) 16.0–19.6 μm, ornamented by crowded cylindric warts measuring 1.8–3 μm high and 2–3 μm wide. This taxon appears to be preferentially or exclusively associated with species of Pinus. When young, due to its yellowish or brownish color, G. pinicola can be confused with young G. arenaria or G. thaxteri, but G. arenaria usually has peridial hairs, while the latter has more lobed ascomata when mature. It is often misidentified as G. pseudobalsleyi due to its rough, warty peridium. In addition, unlike G. pinicola, these two species are not typically collected under Pinus spp. Genea brunneocarpa is another brownish and single-chambered but is not commonly found with Pinus spp., rarely has ascomata that are depressed, and has more pointed peridial warts with prominent and scattered spore ornamentation. Genea zamorana presents reddish tones in the innermost rows of the pseudoparenchymatic peridium layer, a feature not observed in any other species of genus Genea.
Odor remains unremarkable
Compared to the type specimen, G. pinicola from Hungary was ornamented with cylindrical warts measuring 1.8–3 μm high and 2–3 μm wide. Phylogenetic analysis also put our specimen nested within the G. pinicola clade, supported by maximal statistical values at both terminal and backbone nodes (BS: 100%).
Material studied:
ZB-6097, Csővár, Pest County, Hungary, 47° 48′ 45.796” N, 19° 19′ 24.244” E, 4 February 2013, leg. Szilvia Farkas, under Pinus nigra, Genbank accession number PX756463 (ITS).

5. Ecology

5.1. Habitat

From an ecological point of view, we have noticed both similarities and differences. According to the Pluto F biodiversity platform [66], which consists of most of the major European Genea species data, the ascomata of G. pseudobalsleyi were usually found at altitudes of 15–120 m, and G. hispidula at 280–425 m, G. lespiaultii at 25–41 m, followed by G. fragrans at 375–625 m above sea level, reflecting differential altitudinal distributions among European Genea species, with G. lespiaultii occurring predominantly in lowland habitats and G. fragrans at higher elevations.
From our herbarial data, the highest number of Genea species (five) were found in Carici pilosae-Carpinetum vegetation type, followed by Melampyro bihariensi-Carpinetum with three species, Quercetum petraeae-cerris, Querco roboris-Carpinetum betuli, Castanea sativa plantation and Quercetum roboris with two species each, respectively (Figure 6). Among species, G. pseudobalsleyi was recorded most frequently in hilly areas across eight vegetation types, followed by G. fragrans and G. pseudoverrucosa across six vegetation types. As for G. szemereiensis, mostly found in the lowlands, the dominant putative host plants are Q. robur and Carpinus betulus, while for the most closely related species, G. pseudobalsleyi, it is known to be more frequently associated with Quercus petraea as well as C. betulus (in some cases it has also been found in Knautio drymeiae-Ulmetum and Castania sativa plantation). Genea pinicola, on the other hand, is commonly found in symbiosis with Pinus spp. in calcareous soils.

5.2. Soil Characteristics

The PCA of soil data from Genea habitats in Hungary showed 34.9% of the variance (PC1 = 25.4%, PC2 = 15.8%) and revealed a broader edaphic structure (Figure 7a). PC1 exhibited a positive association with salinity, total nitrogen, calcium carbonate, phosphorus pentoxide, humus, and pH (KCl), while it demonstrated a negative association with pH (H2O), manganese, zinc, and potassium oxide. PC2 was predominantly associated with sulfur, magnesium, copper, and sodium. The ordination suggested partial separation of certain Genea species along these soil gradients (Figure 7a). PERMANOVA revealed a marginal but non-significant difference in multivariate soil physicochemical composition among Genea species groups (F = 1.356, R2 = 0.100, p = 0.093). The homogeneity of the multivariate dispersion test was not significant (betadisper: F = 1.502, p = 0.211), indicating no significant difference in within-group dispersion among species. However, two species pairs differed significantly after Benjamini–Hochberg correction: G. fragrans vs. G. pseudobalsleyi (adjusted p = 0.0375) and G. pseudobalsleyi vs. G. pseudoverrucosa (adjusted p = 0.015) (Supplementary File S1, Table S4_a).
The PCA plot (Figure 7b) based on the data from the Pluto F biodiversity platform [66] demonstrated 43.8% of the variance on the first two axes (PC1 = 25.4%, PC2 = 18.4%) and is formed primarily by a base-cation signal, with Ca loading strongly on positive PC1, while K and Mg loaded positively on PC2. PERMANOVA indicated a significant difference in multivariate soil physicochemical composition among Genea species groups in the Pluto F dataset (F = 3.847, R2 = 0.221, p = 0.001). The homogeneity of the multivariate dispersion test was also significant (betadisper: F = 2.443, p = 0.030), indicating unequal within-group dispersion among species. Four pairs differed significantly after Benjamini–Hochberg correction: G. darii vs. G. hispidula (adjusted p = 0.0225), G. darii vs. G. verrucosa (adjusted p = 0.015), G. hispidula vs. G. lespiaultii/sphaerica (adjusted p = 0.015), and G. lespiaultii/sphaerica vs. G. verrucosa (adjusted p = 0.015) (Supplementary File S1, Table S4_b).
In both cases, PCA and PERMANOVA revealed that most Genea species had overlapping soil physicochemical conditions. This indicated that soil variables significantly influence the distribution of Genea species, potentially leading to the establishment of specific ecological niches. Previous studies [68,69,70] have shown that the abundance of individual taxa correlated with the levels of one or more specific soil factors (pH, salinity, mineral composition, and depth).
  • Key to European Genea species
1a. Ascomata black or reddish, often regularly lobed……………………………………2
1b. Ascomata black or reddish, generally irregularly lobed, ………….…………………3
1c. Ascomata brown, tan, yellowish or reddish……………………………………………7
2a. Spores ornamented with flat warts…………………………….………… G. lespiaultii
2b. Spores ornamented with small irregular warts…………………………… G. lobulata
2c. Spores ornamented with hemispherical warts………………….………… G. sphaerica
3a. Spores > 32 μm long with anvil-shaped warts (see also 6g) …………………………4
3b. Spores < 32 μm long ……………………………………………………………………5
4a. Irregularly lobed, tiny, blunt warts, small tuft of hyphae……….……… G. fragrans
4b. Moderately lobed, polygonal warts, abundant tuft of hyphae………… G. fageticola
5a. Truncated sporal warts > 2 μm broad ……………………………G. pseudoverrucosa
5b. Sporal warts typically > 2 μm broad, covered with conical and irregular and occasionally truncated ornamentation………………………………………… G. szemereiensis
5c. Sporal warts < 2 μm broad, with small polygonal peridial warts……G. pseudobalsleyi
5d. Spores with a combination of irregular, pointed or conical warts…….…………… 6
6a. Sporal warts > 3 μm high, either fang-like or molarlike …………………G. dentata
6b. Sporal warts pyramidal/pointed, irregularly lobed with truncated spore ornaments 3–5 (–6) μm high………………………………………………………G. cephalonicae
6c. Subglobose spores, mucronate warts with tooth-like sporal ornaments………………….…………………………………………….………….… G. coronata
6d. Irregular, pointed, conical spores, fruiting in spring or summer………. G.verrucosa
6e. Irregular, rounded or pointed spores, fruiting in winter………………. G. subbaetica
6f. Conical sporal warts < 32 μm long……………………………………… G. compressa
6g. Conical sporal warts > 32 μm long……………………………………. G. vagans
7a. Spores > 36 μm long, obtuse, roundish warts…………………………… G. hispidula
7b. Spores < 30 μm long, trunco-conical or almost cubical warts, reddish color layer between external
pseudoparenchymatic layer and an inner prosenchymatic layer………… G. zamorana
7c. Spores > 21 μm long ……………………………….………………….………………… 8
8a. Associated with Pinus spp., with medium truncated warts ……………. G. pinicola
8b. Associated with Quercus spp., with medium truncated warts …… G. brunneocarpa
8c. Large hemispherical sporal warts ……………………………………………G. oxygala
8d. Small pointed sporal warts ………………………….…………….…… G. tuberculata

6. Discussion

Compared to other fungal taxa, the taxonomy of the genus Genea has not been frequently modified or new species introduced or revised until recently [71]. Here we describe a new species and revise the taxonomy of Genea based on our morphological, molecular and ecological data. In this study, we used genetic markers for the LSU and specifically for the ITS region [72], as it is the dominant fungal barcode, even though it comes with high variability [73]. Although we successfully used LSU in this study, it is generally more effective for resolving higher taxonomic levels because of its amplification bias toward Ascomycota [74]. We noticed robust and statistically significant differences, especially in spore length between the new species and the closely related species during the revision, which proved that in case of a complex genus like Genea, spore morphology remains a more powerful tool in diagnosis and discrimination for species. Preliminary FT-IR spectroscopic data also provided supplementary chemical evidence supporting the morphological distinction of G. szemereiensis. Despite excluding formal chemometric classification, the spectral profile of G. szemereiensis demonstrated notable differences from its closely related species in the fingerprint and high-wavenumber regions, consistent with its proposed taxonomic arrangement. Moreover, in this study, both mPTP [75] and bPTP [76] analyses placed G. szemereiensis as a distinct species-level lineage; however, caution should be applied while applying species delimitation methods, as they can be imperfect representations of biological systems [77]. We also noticed some sequences from GenBank and Unite databases clustering with G. szemereiensis, without any formal description or, in some cases, proper identification, which were also added in the phylogenetic tree for transparency. The classic G. pseudobalsleyi species is not densely covered with ornamentation [3]; however, the new species, i.e., G. szemereiensis, which is closely related to G. pseudobalsleyi, was observed to be densely covered with ornamentation. We also encountered some cryptic or transient species that could be differentiated molecularly but could not be segregated based on their morphological parameters. Hence, in future works, standardized preservation of the specimens should be considered, as improper preservation can lead to misidentification. Some specimens, which were not preserved or could not be identified morphologically as they had disintegrated, were sent for NGS, which yielded better results and will be explored in upcoming works. Based on our new identification key, the results of the revision of previously reported Genea materials [78,79] did not match any similar species when compared with the new species reported here, indicating it as a novel species.
In the beginning of this study, we highlighted one key point, and that was the ability of Genea to form ectomycorrhiza (ECM). However, the inclusion of Genea in ECM surveys is largely missing, probably due to the preference of old growth or undisturbed habitats [80] and the difficulty of locating hypogeous fungi and the Ascomycota [42]. In Hungary, the Genea species were never found in young forest habitats or in young truffle plantations [81], which confirms that Genea species are late-stage mycorrhizal fungi.
Until now, two mycorrhizae of Genea have been published. These are G. hispidula Berk. & Br. + Fagus and G. verrucosa Vitt. + Quercus [82]. Fruit bodies of the later ectomycorrhiza are deposited under the ZB-1057. Based on our new identification key, the species was verified to be Genea verrucosa, but the NGS sequencing resulted in the report of a hyperparasitic fungus, Hypomyces stephanomatis, which is reported from Humaria (a closely related genus of Genea) [83].
During an ongoing metagenomic survey of the ECM community of Quercus rubra (L.) in Hungary, Genea hispidula was detected on the ECM-colonized root tips and soil under Quercus rubra in a rather acidic habitat (pH 3.45) (Péter B., unpublished data). This is the first report from the metagenomic analysis of this species from Hungary. G. hispidula and other hypogeous species with low pH soil preferences could remain undetected in Hungary, as the acidic forests are out of the scope of truffle collectors.
Ecologically, in this study, Carici pilosae–Carpinetum and Melampyro bihariensei–Carpinetum exhibited the highest Genea richness, indicating that these forest types provide particularly favorable habitat conditions. The PCA data from the Pluto-F platform showed less distinct separation among Genea species, possibly indicating greater overlap in habitat preferences at the continental scale, whereas the PCA based on our herbarial data showed comparatively clearer separation of Genea species along soil-chemical gradients, suggesting possible edaphic differentiation at the local scale. These contrasting ordination patterns are likely due to differences in sample size, geographic scale, and environmental data standardization. The presence of both widespread and restricted species within a well-supported phylogeny reflects the complex evolutionary dynamics, including dispersal, ecological specialization, and cryptic speciation.
In conclusion, biogeographically, Hungary emerges as a significant center of diversity for hypogeous Pezizaceae, harboring elements of different biomes and distributed species from different regions. The position of the Carpathian Basin at the confluence of several European climatic and vegetation zones may contribute to the observed species richness by providing a heterogeneous landscape capable of supporting taxa with differing ecological requirements. Based on the genus revision, G. szemereiensis was concluded as a novel species based on genetic cohesiveness and stable topology from the phylogenetic analyses. In the process, we contribute to the addition of two new species reported from Hungary, G. szemereiensis and G. pinicola, to the list of previously published ones from Hungary (G. fragrans, G. lespiaultii-sphaerica, G. verrucosa, G. pseudoverrucosa, G. pseudobalsleyi, G. cf. subbaetica, G. hispidula, G. compressa).

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/d18060360/s1, File S1: Table S1. List of species, herbarium vouchers, GenBank/UNITE accession numbers for ITS and LSU sequences, and origin of materials analyzed in the phylogenetic study. Table S2. List of materials involved in morphological studies. Table S3. Comparative micromorphological traits of the ascospores of the most frequently occurring Genea species from Central Europe. Table S4. Pairwise PERMANOVA comparisons of multivariate soil physicochemical composition among Genea species (a) from herbarial data (Table S4_a) and (b) the Pluto F biodiversity platform based on standardized soil variables (Table S4_b). Figure S1. SEM images of Genea species. Figure S2. Phylogram generated from the Bayesian inference analysis of Genea sequences based on internal transcribed spacer (ITS) and nuclear large subunit ribosomal DNA (LSU) sequence data. Figure S3. Phylogenetic reconstructions of Genea based on maximum likelihood analysis of two loci individually, ITS and LSU. Figure S4. mPTP analysis for the genus Genea. Figure S5. Single PTP analyses for the genus Genea based on Bayesian and maximum likelihood topologies. Figure S6. FT-IR spectra of Genea species. File S2: List of specimens used in multilocus phylogenetic (ITS, LSU) analysis and their GenBank accession numbers. File S3: List of specimens used in the single-locus phylogenetic (ITS) analysis and their GenBank accession numbers.

Author Contributions

S.C. and Z.B. contributed to the concept and methodology. Z.B. and M.B. participated in the field collections. Morphological measurements were performed by S.C., S.A.T. and Z.B.; morphometric analysis and statistics were done by S.C.; SEM photographs were taken by A.K., Z.K. and S.C.; FTIR measurements and interpretation were finalized by M.B., A.K. and S.C.; S.C. and S.A.T. carried out the DNA extraction, primer selection and PCR. The phylogenetic tree was constructed by B.D.P., A.A.H. and S.C.; graphs, illustrations, data preparation and writing of the ecology were performed by G.C.W. and S.C. The overall manuscript was organized, written and followed up by S.C. This work was supervised by Z.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Ministry of Culture and Innovation of Hungary through the National Research, Development and Innovation Fund, under the KDP-2023 funding scheme (grant number C2268483).

Institutional Review Board Statement

Not Applicable.

Informed Consent Statement

Not Applicable.

Data Availability Statement

The voucher specimen ZB-6043 (BP 113476/Barcode: HNHM-MYC070988) is submitted to the Magyar Természettudományi Múzeum. (Hungarian Natural History Museum) and registered in MycoBank. All sequence data generated for this study can be accessed via GenBank: https://www.ncbi.nlm.nih.gov/genbank/ (accessed on 5 June 2026). All alignments and additional data have been submitted as Supplementary Materials. Any raw data will be made available with a reasonable request.

Acknowledgments

The authors would like to express their gratitude to the members of EMSzE and other truffle collectors, who helped in the collection of the samples. Furthermore, we express our thanks to Biomi Ltd. for the sequencing of our samples.

Conflicts of Interest

The authors declare no competing interests.

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Diversity 18 00360 g001
Figure 1. Differences in ascospore length (a) and width (b) were determined using one-way Welch’s ANOVA, followed by Dunnett’s T3 post hoc test for multiple comparisons. Different lowercase letters indicate significant difference (p < 0.05).
Figure 1. Differences in ascospore length (a) and width (b) were determined using one-way Welch’s ANOVA, followed by Dunnett’s T3 post hoc test for multiple comparisons. Different lowercase letters indicate significant difference (p < 0.05).
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Figure 2. Phylogram generated from maximum likelihood (ML) analyses of Genea sequences based on internal transcribed spacer (ITS) and nuclear large subunit ribosomal DNA (LSU) sequence data, with H. hemisphaerica and H. cazaresii as outgroups. ML bootstrap values (BS) were obtained from 1000 replicates, and values > 70% were indicated on the branches. Scale bar 0.03 indicates expected nucleotide changes per site. G. szemereiensis and G. pinicola are marked in red. Asterisks (*) denote newly generated sequences.
Figure 2. Phylogram generated from maximum likelihood (ML) analyses of Genea sequences based on internal transcribed spacer (ITS) and nuclear large subunit ribosomal DNA (LSU) sequence data, with H. hemisphaerica and H. cazaresii as outgroups. ML bootstrap values (BS) were obtained from 1000 replicates, and values > 70% were indicated on the branches. Scale bar 0.03 indicates expected nucleotide changes per site. G. szemereiensis and G. pinicola are marked in red. Asterisks (*) denote newly generated sequences.
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Figure 3. Phylogram generated from Bayesian inference (BI) analyses of Genea sequences based on internal transcribed spacer (ITS) sequences within the G. balsleyi-pseudobalsleyi complex. Bayesian posterior probabilities (BPP > 0.95) are mentioned on the branches, and the scale bar 0.015 represents expected nucleotide changes per site. G. szemereiensis is marked in bold.
Figure 3. Phylogram generated from Bayesian inference (BI) analyses of Genea sequences based on internal transcribed spacer (ITS) sequences within the G. balsleyi-pseudobalsleyi complex. Bayesian posterior probabilities (BPP > 0.95) are mentioned on the branches, and the scale bar 0.015 represents expected nucleotide changes per site. G. szemereiensis is marked in bold.
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Figure 4. Genea szemereiensis. (a,b) Ascomata; (c) microscopic cross-section of ascomata (DIC); (d) Septae of paraphyses (DIC) from macerated epithecium layer; (e) single ascospore; (f) spores contained within the ascus (DIC); (g,h) SEM micrographs of spores; scale bars 1 mm (a,b), 100 µm (c) and 10 µm (d–h).
Figure 4. Genea szemereiensis. (a,b) Ascomata; (c) microscopic cross-section of ascomata (DIC); (d) Septae of paraphyses (DIC) from macerated epithecium layer; (e) single ascospore; (f) spores contained within the ascus (DIC); (g,h) SEM micrographs of spores; scale bars 1 mm (a,b), 100 µm (c) and 10 µm (d–h).
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Figure 5. Genea pinicola. (a,b) Ascomata; (c) microscopic cross-section of ascomata (DIC); (d) Septae of paraphyses (DIC) from macerated epithecium layer; (e) single ascospore; (f) spores contained within the ascus (DIC); (g,h) SEM micrographs of spores; scale bars 1 mm (a,b), 100 µm (c) and 10 µm (d–h).
Figure 5. Genea pinicola. (a,b) Ascomata; (c) microscopic cross-section of ascomata (DIC); (d) Septae of paraphyses (DIC) from macerated epithecium layer; (e) single ascospore; (f) spores contained within the ascus (DIC); (g,h) SEM micrographs of spores; scale bars 1 mm (a,b), 100 µm (c) and 10 µm (d–h).
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Figure 6. Vegetational associations of Genea species occurrences based on our database identified by Prof. Simon Tibor (2000) [67].
Figure 6. Vegetational associations of Genea species occurrences based on our database identified by Prof. Simon Tibor (2000) [67].
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Figure 7. Principal Component Analysis (PCA) biplot from (a) current study soil data and (b) Pluto F biodiversity platform, showing relationships between soil chemical properties and Genea species distribution. Arrows indicate the direction and magnitude of soil variables, points represent sampling sites associated with each species, and ellipses represent 68% confidence intervals of species distributions.
Figure 7. Principal Component Analysis (PCA) biplot from (a) current study soil data and (b) Pluto F biodiversity platform, showing relationships between soil chemical properties and Genea species distribution. Arrows indicate the direction and magnitude of soil variables, points represent sampling sites associated with each species, and ellipses represent 68% confidence intervals of species distributions.
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Chakraborty, S.; Tirpude, S.A.; Péter, B.D.; Walle, G.C.; Habtemariam, A.A.; Kedves, A.; Balogh, M.; Kónya, Z.; Bratek, Z. Taxonomy, Phylogeny and Ecological Assessment of the Truffle Genus Genea in Central Europe with a New Species and a New Record. Diversity 2026, 18, 360. https://doi.org/10.3390/d18060360

AMA Style

Chakraborty S, Tirpude SA, Péter BD, Walle GC, Habtemariam AA, Kedves A, Balogh M, Kónya Z, Bratek Z. Taxonomy, Phylogeny and Ecological Assessment of the Truffle Genus Genea in Central Europe with a New Species and a New Record. Diversity. 2026; 18(6):360. https://doi.org/10.3390/d18060360

Chicago/Turabian Style

Chakraborty, Swagata, Shruti Anand Tirpude, Balázs Domonkos Péter, Getnet Chekole Walle, Akale Assamere Habtemariam, Alfonz Kedves, Máté Balogh, Zoltán Kónya, and Zoltán Bratek. 2026. "Taxonomy, Phylogeny and Ecological Assessment of the Truffle Genus Genea in Central Europe with a New Species and a New Record" Diversity 18, no. 6: 360. https://doi.org/10.3390/d18060360

APA Style

Chakraborty, S., Tirpude, S. A., Péter, B. D., Walle, G. C., Habtemariam, A. A., Kedves, A., Balogh, M., Kónya, Z., & Bratek, Z. (2026). Taxonomy, Phylogeny and Ecological Assessment of the Truffle Genus Genea in Central Europe with a New Species and a New Record. Diversity, 18(6), 360. https://doi.org/10.3390/d18060360

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