Abstract

Hebeloma species are ectomycorrhizal fungi that are widely distributed in nature and grow in a wide variety of habitats. Hebeloma spp. are difficult to identify and distinguish due to high morphological similarity between species. In this study, two newly recorded Hebeloma (Fr.) P. Kumm. species (H. minus Bruchet and H. rostratum Beker, Vesterh. & U. Eberh.) from Türkiye were described and photographed. Specimens were identified using morphological and molecular methods. Phylogenies based on the internal transcribed spacer (ITS) and translation elongation factor 1-alpha (TEF1-α) regions of the genome were constructed employing the Maximum-Likelihood method. In conclusion, a technique that combines morphological and molecular data provides a robust approach to assessing the taxonomic status of Hebeloma spp. Descriptions and photographs of the species are also presented.

Introduction

Hebeloma (Fr.) P. Kumm. (Basidiomycota, Hymenogastraceae) is a fungal genus growing widely over varying habitats and forming symbiotic relationships with various tree species (Cripps et al., 2019). Currently, more than 500 Hebeloma species have been recognized globally (https://www.indexfungorum.org), including a few undocumented ones. Beker et al. (2016) conducted a worldwide study and documented this genus comprehensively. In the Hebeloma monograph published by Beker et al. (2016), 84 species were recognized based on morphological and molecular characters. Additionally, 51 species were identified in other studies conducted by the Beker group (Cripps et al., 2019; Eberhardt et al., 2020; Eberhardt et al., 2021a, 2021b; Eberhardt et al., 2022a, 2022b, 2022c). In total, 135 species have been recorded globally using morpho-molecular analysis, of which 35 have been identified in Türkiye (Dizkırıcı et al., 2019, 2022; Sesli et al., 2020; Acar et al., 2021, 2022, 2024; Solak & Türkoğlu, 2022; Lambert et al., 2024). Among them, only eight have been studied using molecular data (Dizkırıcı et al., 2019, 2022; Acar et al., 2021, 2022, 2024; Lambert et al., 2024).

Hebeloma species are morphologically characterized by a bicolored convex pileus, mostly yellowish-brown lamellae, a veil, and abundant cheilocystidia (Eberhardt et al., 2015, 2021b, 2022b; Beker et al., 2016). However, as identifying the species within this genus remains challenging, infrageneric classification requires both morphological and molecular data (Beker et al., 2016). The internal transcribed spacer (ITS) region is considered one of the most vital DNA barcodes applicable for species-level identification in fungal taxonomy (Schoch et al., 2012). It is commonly used in phylogenetic studies due to easy amplification, the availability of vast amounts of related data in the GenBank database, and a distinct barcode gap for species determination (Schoch et al., 2012). It can be utilized alone or in combination with protein-coding genes like the translation elongation factor 1-alpha (TEF1-α) to improve precision in the molecular identification of fungi (Tekpinar & Kalmer, 2019).

This study aimed to report and document two new records, H. minus Bruchet and H. rostratum Beker, Vesterh. & U. Eberh. from Türkiye, identified based on morphological features and molecular data. It also evaluates the phylogenetic affinities of the two species.

Materials and Methods

Fungal Materials and Morphological Observations

The Hebeloma specimens were collected from Hakkâri province of Türkiye. The samples were photographed in the field using an EOS 60D camera (Canon, Tokyo, Japan) equipped with an AT-X 100 mm macro lens (Tokina, Tokyo, Japan). The macroscopic characteristics were determined based on field notes and photographs. For microscopic examinations, thin cuttings from the lamellae and basidiomata surfaces were obtained with razor blades. They were then treated with distilled water and Melzer’s reagent. The material was microscopically examined underwater to count basidiospores, cystidia, and basidia, and to determine other vital features, but in Melzer’s solution to assay the dextrinoid reactions (Vesterholt, 2005; Beker et al., 2016). At least 50 spores, 40 basidia, and cheilocystidia were measured from each specimen of each recorded species. Observations were made using a DM500 research microscope (Leica Microsystems), and images were captured utilizing the Leica Application Suite (version 3.4.0). The length-width ratio of the basidiospores is referred to as “Q” in the text. The dextrinoid reaction of spores in the Melzer’s reagent (Atom scientific) is mentioned as “D.” The loosening of the spore perisporium in Melzer’s reagent is noted as “P.” The ornamentation of the spore is recorded as “O.” All materials studied were preserved in the Fungarium of the Van Yüzüncü Yıl University, Van, Türkiye.

DNA Isolation and Sequencing

CTAB Doyle & Doyle, 1987). The ITS and TEF1-α regions were polymerase chain reaction (PCR)-amplified using the primer sets N-nc18S10/C26A (Wen & Zimmer, 1996) and EF1-983F/EF1-1567R (Rehner & Buckley, 2005). PCR was performed in a total volume of 25 µL, containing genomic DNA (10 ng/µL), 10X Buffer, MgCl₂ (25 mM), dNTP mix (10 mM), primer pair (10 µM), Taq Pol (5U/µL), and sterile water. The thermocycling conditions included, an initial denaturation at 94℃ for 3 min, followed by 35 cycles of denaturation at 95℃ for 1 min, annealing at 51℃ (52℃ for TEF1-α) for 35 s, and extension at 72℃ for 40 s, with a final extension at 72℃ for 5 min. The amplicons were separated using 1% TAE agarose gel and stained with Gelred dye. The positive reactions were bidirectionally sequenced using the same primer pairs on an ABI 3730XL automated sequencer at BM Labosis Inc., Ankara, Türkiye. The sequences generated were submitted to the GenBank, and their accession numbers are listed in Table 1.

Phylogenetic Analysis

The chromatograms were visualized using Finch TV (http://www.geospiza.com). Consensus sequences were generated and utilized for BLAST alignment against the sequences available in the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/). The novel sequences were submitted to the NCBI and related taxa identified were downloaded to establish the phylogenetic relationships (Table 1). H. mesophaeum (Pers.) Quél. and H. sacchariolens Quél. were chosen as the outgroup taxa. All sequences were aligned using CLUSTALW (Thompson et al., 1994) and refined with the Mesquite v3.70 software (Maddison & Maddison, 2021).

Phylogenetic analyses utilized a concatenated ITS/TEF1-α dataset, and the Maximum-Likelihood (ML) method was applied. The best substitution model for each partition was calculated via the Modelfinder algorithm (Kalyaanamoorthy et al., 2017). ML analysis was performed with IQ-TREE v.1.6.12 (Nguyen et al., 2015) employing the K2P + R2 model. Branch support was assessed through 1,000 replicates of the ultrafast bootstrap (Hoang et al., 2018) and Shimodaira-Hasegawa-like approximate likelihood ratio tests (Guindon et al., 2010). The phylograms were visualized using Figtree v1.4.3 (Rambaut 2016).

Results

Taxonomy

Descriptions, locations, collection dates, fungarium numbers, and illustrations of the identified taxa are provided below.

Hymenogastraceae Vittad.

Hebeloma sect. Denudata subsect. Crustliniformia

H. minus Bruchet, Bull. Mens. Soc. Linn. Lyon 39 (suppl. 6): 126 (1970), (Figure 1)

Description: Pileus: 10-25(30) mm, shape convex to umbonate; generally bicolored, yellow-brown or dark brown in center, margin smooth, sometimes curly. Lamellae: emarginate, occasionally adnate, L = 26-40. Cortina absent. Stipe: 10-30(40) × 2-8 mm, generally cylindrical, clavate, often pruinose. Odor: absent or slightly radish-like. Basidiospores: (8.6)9-15 × 5-7(7.9) µm (n = 50), on average 12.8 × 6.4 ± 0.9 (length), ± 0.4 (width); Q = 1.6-1.91, amygdaliform, brown or yellow-brown, ornamentation distinct (O2-O3), no loosening perispore (P0-P1), slightly dextrinoid (D1-D2). Basidia: 27-40 × 7-15 µm (n = 40), four-spored. Cheilocystidia: 35-70(80) × 7(8)-10(14) × 3-7 × 2-7(8) µm (n = 40), on average 40-62 × 7-10 (apex) × 3-5 (middle) × 3-6 (base) µm, usually clavate-stipitate, often capitate-stipitate, contents yellow. Caulocystidia: like cheilocystidia, up to 100 µm.

Specimen examined: Hakkâri, Yüksekova, Adaklı Village, meadow area, 37°31′34″N and 44°10′12″E, elev. 1,870 m a.s.l., 03.11.2012; VANF4, leg. S. Kesici. 37°31′39″N and 44°10′04″E, elev. 1,872 m a.s.l., 03.11.2012; VANF5, leg. S. Kesici. 37°31′35″N and 44°10′11″E, elev. 1,868 m a.s.l., 03.11.2012; VANF16, leg. S. Kesici.

Notes: H. minus is classified within the Denudata section with its clavate cheilocystidia (Beker et al., 2016). The species is distinguished from other members of the section by its mostly ornamented basidiospores. The monograph by Beker et al. (2016) states that the species prefers Arctic tundra habitats with mossy soils. Eastern Anatolia and its environs are included in the Irano-Turanian phytogeographical region of Türkiye. The Irano-Anatolian region is a mountainous area, and Parolly (2004) classified it as high mountain grasslands. H. minus specimens have also been found in the high mountain grassland areas of Türkiye.

Hebeloma sect. Denudata subsect. Echinospora 

H. rostratum Beker, Vesterh. & U. Eberh., Fungal Biol. 120: 96 (2015), (Figure 2)

Description: Pileus: 16-35(40) mm, shape convex, sometimes umbonate, generally bicolored; yellow-brown, cream-brown, cream, dark brown in center, margin curly, sometimes smooth. Lamellae: generally emarginate, adnate, L = 30-65. Cortina absent. Stipe: 20-45(50) × 2-6 mm, cylindrical at the base, clavate, floccose at the apex. Odor: absent or slightly radish-like. Basidiospores: 6-13(14.6) × (5.5)6-8(8.7) μm (n = 50), on average 11.2 × 6.7 ± 0.7 (length), ± 0.33 (width); Q = 1.62-1.95, amygdaliform, yellow-brown; strongly ornamented (O3-O4), loosening perisporium (P2-P3), strongly dextrinoid (D3-D4). Basidia: 24−45 × 6−10 μm (n = 40), four-spored. Cheilocystidia: 40-60(90) × 3(4)-7 × 3-6(8) × 4-10(12) μm (n = 40), on average 44-65 × 5-7 (apex) × 4-6 (middle) × 6-9 (base) μm, often clavate-lageniform or clavate-ventricose, bifurcate, rostrate, contents yellow. Caulocystidia: like cheilocystidia, larger, up to 120 μm.

Specimen examined: Hakkâri, Yüksekova, Gürdere Village, under Populus spp., 37°29′48″N and 44°12′53″E, elev. 1,962 m a.s.l., 06.11.2012; VANF50, leg. S. Kesici. 37°30′08″N and 44°12′50″E, elev. 1,894 m a.s.l., 03.11.2013; VANF178, leg. S. Kesici.

Notes: This species of the Denudata section is distinguished from other members by its rostrate-shaped cheilocystidia and cheilocystidia apex measuring up to 98 µm (Beker et al., 2016). According to the monograph of Beker et al. (2016), H. rostratum prefers grassy or marshy soils. The most recorded associations of this species were with Populus spp. (Beker et al., 2016). Turkish specimens have also been found under Populus spp.

Phylogenetic Analyses

The DNA sequences of the ITS and TEF1-α genomic regions of H. minus and H. rostratum specimens were deposited in the GenBank database. After removing the missing data and ambiguous regions, the final ITS and TEF1-α datasets were 567 and 524 bp long, respectively. In total, 127 and 89 variable sites were identified in the ITS and TEF1-α datasets, respectively. The length of the concatenated sequence was ~1,158 bp, comprising 176 variable sites.

The combined dataset consisting of 38 sequences, including the five studied here and two outgroups, was utilized to construct the phylogenetic tree. It illustrated the phylogenetic relations and taxonomic positions of the species studied (Figure 3). Members of the Denudata section formed a monophyletic clade with robust support (100%). The section was subdivided into two main clusters: the newly recorded H. minus species clustered within clade A (subsect. Crustuliniformia) with reference sequences of the same species with robust support (99%), and H. rostratum grouped closely with its representatives in clade B (subsect. Echinospora) with high support (99%). These results provide strong evidence for the taxonomic classification of both species within the section Denudata with high bootstrap values.

Discussion

The present study provided morphological descriptions of two newly recorded species of the Hebeloma genus from Türkiye. They were identified based on morphological features and then verified using molecular data. After confirmation, the phylogenetic relationships of these species with close relatives within the genus were established.

The species presented in this study were included in the Denudata section, which is the largest within the Hebeloma genus. The most important feature of this section is the cheilocystidia morphology, with swollen apices and constricted midsections. Members of this section generally lack a cortina, and their odor is usually described as radish-like. The lamellae typically have clear droplets. Basidiospores are usually almond-shaped. The spore codes were O1-3; P0-2; and D1-3, on an average. The average spore size was 9−12 × 5−7.5 μm, and the mean Q value was 1.60−2.10. The average length of the cheilocystidia was between 40 and 75 μm (Beker et al., 2016). Eberhardt et al. (2016) recognized four subsections: Crustuliniformia, Clepsydroida, Hiemalia, and Echinospora within this section. Crustuliniformia is characterized by clavate-stipitate and clavate-capitate cheilocystidia. Clavate-stipitate to clavate-ventricose cheilocystidia indicate that a species is from Echinospora (Beker et al., 2016; Eberhardt et al., 2016).

The phylogeny presented in this work confirmed that H. minus belongs to Crustuliniformia. It can be distinguished from other members by its highly ornamented spores (Eberhardt et al., 2016). H. minus occupied a clearly distinct position in the phylogenetic tree and was closely related to H. pallidolabiatum Beker & U. Eberh. These two species resembled one another, but could be distinguished microscopically based on the shape of cheilocystidia and ornamentation of the spores, which were often capitate-stipitate and O2-3 for H. minus but clavate-lageniform and O2 for H. pallidolabiatum (Beker et al., 2016). H. minus was also macroscopically similar to H. alpinum (J. Favre) Bruchet. However, detailed examinations revealed that H. minus had smaller basidiomes with a darker coloration and fewer full-length lamellae, with counts < 40. This was in contrast to H. alpinum, which consistently exhibited ≥ 40 full-length lamellae (Cripps et al., 2019; Eberhardt et al., 2021b).

H. rostratum belongs to Echinospora and is characterized by clavate-ventricose cheilocystidia and amygdaliform-shaped spores (Eberhardt et al., 2016). It draws attention via its rostrate cheilocystidia that distinguish it from other species within the section when examined microscopically. The species was first collected in Italy by Eberhardt et al. (2016) and reported to grow under Salix and Populus spp. H. rostratum occupied a clearly distinct position in the phylogram and was closely related to H. populinum Romagn. The H. rostratum specimens studied can be differentiated based on highly warty spores and cheilocystidia apex with dimensions averaging < 7.5 µm.

Conclusion

Türkiye harbors a noticeable climatic and phytogeographical diversity, supporting a wide variety of vegetation. It is hypothesized that many fungal species are yet to be discovered in this country. The fact that species belonging to Hebeloma have been studied extensively across the globe has enabled the construction of a comprehensive database. It allows us to cluster a newly discovered species into collections with similar characteristics and to compare the collection parameters within the same phylogenetic clade. Considering such a situation, the lack of data for this genus in Türkiye is a serious scientific lacuna. Being aware of this deficiency, we performed diagnostic studies on the samples collected during every field study and accumulated data for Hebeloma. Based on the data collected in the present work, we report two new species from Türkiye and present them to the scientific community.