Three new species and one new record of Scleroderma (Sclerodermataceae, Boletales) from northern Thailand

69 Three new species and one new record of Scleroderma (Sclerodermataceae, Boletales) from northern Thailand Didsanutda Gonkhom1,2 , Phongeun Sysouphanthong2, Marc Stadler3,4 , Naritsada Thongklang1,2 , Kevin D. Hyde2 1 School of Science, Mae Fah Luang University, Chiang Rai, Thailand 2 Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, Thailand 3 Department of Microbial Drugs, Helmholtz Centre for Infection Research (HZI), 38124 Braunschweig, Germany 4 German Centre for Infection Research (DZIF), partner site Hannover/Braunschweig, Inhoffenstrasse 7, 38124 Braunschweig, Germany Corresponding authors: Naritsada Thongklang ([email protected]); Kevin D. Hyde ([email protected]) Copyright: © Didsanutda Gonkhom et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract The genus Scleroderma (Sclerodermataceae) contains gasteroid ectomycorrhizal fungi and is distributed worldwide in temperate and tropical regions. Fresh specimens were collected in Thailand and report three undescribed species and one new record for the country. These species were characterized by photographs of freshly collected basidiomes, and their macroand microscopic features were compared with those of known species of Scleroderma. Additionally, DNA sequence data were generated for four loci, including the nuclear ribosomal internal transcribed spacer region (ITS), the large subunit ribosomal RNA gene (LSU), the translation elongation factor 1-alpha gene (tef1-α), and the second largest subunit of RNA polymerase II (rpb2). A multi-locus phylogeny was constructed to confirm their taxonomic placement. Key words: Basidiomes, Boletales, morphology, taxonomy, three new species Introduction Ectomycorrhizal fungi form symbiotic relationships with the feeder roots of many tree species and other plants that benefit both partners (Charya and Garg 2019). They are distributed worldwide in temperate and tropical regions (Corrales et al. 2018). The ectomycorrhizal fungi include the genus Scleroderma (Ouatiki et al. 2022). Persoon (1801) erected the genus Scleroderma, which was later updated by Guzmán (1970), who proposed infrageneric classifications such as sections Sclerangium, Scleroderma, and Macrospora based on morphology. Modern phylogenetic studies have further refined these groupings (de Menezes Filho et al. 2022; Wu et al. 2023). From early morphology-based classifications to more recent genetic investigations that have improved our understanding of species relationships (Persoon 1801; Guzmán 1970; Wu et al. 2023), the history of Scleroderma demonstrates the evolving dynamics of fungal taxonomy. The evolution from Lycoperdon verrucosum to S. verrucosum illustrates how scientific classification continues to evolve (Persoon 1801). Academic editor: Thorsten Lumbsch Received: 28 May 2025 Accepted: 26 August 2025 Published: 8 October 2025 Citation: Gonkhom D, Sysouphanthong P, Stadler M, Thongklang N, Hyde KD (2025) Three new species and one new record of Scleroderma (Sclerodermataceae, Boletales) from northern Thailand. MycoKeys 123: 69–88. https://doi.org/10.3897/ mycokeys.123.160438 MycoKeys 123: 69–88 (2025) DOI: 10.3897/mycokeys.123.160438 70 MycoKeys 123: 69–88 (2025), DOI: 10.3897/mycokeys.123.160438 Didsanutda Gonkhom et al.: Three new Scleroderma species and a record from northern Thailand There are 206 Scleroderma species listed in Index Fungorum (https://www. indexfungorum.org/Names/Names.asp, accessed on 24 March 2025) and 76 species in Species Fungorum (https://www.speciesfungorum.org/Names/ Names.asp, accessed on 24 March 2025). The genus belongs to the family Sclerodermataceae, order Boletales, and class Agaricomycetes (Binder and Hibbett 2006). Scleroderma species have traditionally been segregated according to the morphology of the basidiomata and the surface of the peridium, the type of dehiscence of the peridium, the color of the gleba, and the ornamentation of their basidiospores (de Menezes Filho et al. 2022). Moreover, the thickness and scaliness of the peridium, the presence of stalks of the basidiome, and the form of the stipe have occasionally been used to distinguish species in the genus (Raut et al. 2020). Although regions such as Europe and America are relatively well studied in terms of Scleroderma taxonomy, data are lacking for tropical Africa (Sanon et al. 1997) and Asia (Farmer and Sylvia 1998; Sims et al. 1999). In Thailand, 11 species have been reported, namely S. areolatum, S. aurantium, S. bovista, S. cepa, S. citrinum, S. dictyosporum, S. flavidum, S. lycoperdoides, S. polyrhizum, S. sinnamariense, and S. verrucosum, based on morphological characteristics (Chandrasrikul et al. 2011) and molecular analysis of S. suthepense (Kumla et al. 2013). In our study, our aim was to describe three new species and a new record of Scleroderma from northern Thailand, based on macroand microscopic characteristics and molecular phylogenetic methods. Materials and methods Sample collection Fresh basidiomes of Scleroderma were collected during the rainy season from May to June 2019 in Chiang Mai and Chiang Rai provinces. A total of 21 specimens were collected, including representatives of three new species. The specimens were dried in an oven at 45 °C for at least 24 hr and stored at room temperature. They are deposited at Mae Fah Luang University Fungarium (MFLU Fungarium). Morphological analysis Fresh basidiomata were described for macrocharacters, photographed in the field, and tested for macrochemical reactions (color reactions) of the peridium with 5% potassium hydroxide (KOH). Their size and color were recorded, and color was compared with the Methuen Handbook of Color (Kornerup and Wanscher 1981). For observation of microcharacters, dried samples were sectioned with razor blades, mounted on glass slides, and rehydrated with 5% KOH (w/v). The sizes and shapes of the microstructures, including hyphae, basidia, and basidiospores, were examined under a microscope (Nikon DS-Ri2). At least two specimens of each species were measured, and each characteristic was measured in at least 50 replicates. Spore ornamentation was also examined. 71 MycoKeys 123: 69–88 (2025), DOI: 10.3897/mycokeys.123.160438 Didsanutda Gonkhom et al.: Three new Scleroderma species and a record from northern Thailand DNA extraction, PCR amplification, and sequencing Genomic DNA was extracted from 100 mg of the fruiting body using the Biospin Fungus Genomic DNA Extraction Kit (Bioer Technology, Hangzhou, China) following the manufacturer’s protocol. The polymerase chain reaction (PCR) was used to amplify the internal transcribed spacer (ITS), the 28S large subunit region of ribosomal DNA (LSU), the second largest subunit of RNA polymerase II (rpb2), and the translation elongation factor 1-alpha (tef1-α). The primer pairs used were: ITS1-F and ITS4 for ITS (White et al. 1990; Gardes and Bruns 1993), LR0R and LR5 for LSU (Vilgalys and Hester 1990; White et al. 1990), RPB2-6F and RPB2-7cR for rpb2 (Rehner and Buckley 2005), and EF1-983F and EF1-1567R for tef1-α (Rehner and Buckley 2005). The PCR cycling conditions for ITS, LSU, rpb2, and tef1-α were: 3 min at 94 °C; 35 cycles of 30 s at 94 °C, 30 s at 52 °C, and 1 min at 72 °C; followed by 10 min at 72 °C. For tef1-α, the program was: 5 min at 95 °C; 35 cycles of 1 min at 94 °C, 2 min at 52 °C, and 1.5 min at 72 °C; followed by 10 min at 72 °C. Sequencing of PCR-amplified products in both directions was performed by Sangon Biological Engineering Technology and Services (Shanghai). Alignment and phylogenetic analysis Phylogenetic analysis and sequence divergence were used to determine the relationship of the newly discovered taxon to other Scleroderma species. In the phylogenetic analysis, Scleroderma species from broader geographic regions were included for comparison. BioEdit Sequence Alignment Editor version 7.0.9.0 was used to verify the ITS, LSU, rpb2, and tef1-α sequences, and SeqMan (DNAstar, Madison, WI, USA) was used to assemble the sequences. The database of the National Center for Biotechnology Information (NCBI) (http://www. ncbi.nlm.nih.gov/genbank/) was searched against each sequence using the Basic Local Alignment Search Tool (BLAST) to ensure that it belonged to the correct genus and was not contaminated and to identify the closest matches. A GenBank BLAST search was performed to check for similarity between the newly generated sequences. In total, 82 sequences of several Scleroderma species from various regions, including our collections, were obtained (Table 1). Pisolithus aurantioscabrosus, a close relative of Scleroderma (Martin et al. 2002; Wilson et al. 2012), was chosen as an outgroup. Alignments were performed using MAFFT v. 7.11 (https://mafft.cbrc.jp/alignment/software/, accessed on 23 December 2023), and all alignments were trimmed separately using TrimAl to eliminate ambiguously aligned positions (Capella-Gutiérrez et al. 2009). The character sets included 78 collections and 676 characters (including gaps) from ITS, 20 collections and 1402 characters from LSU, 20 collections and 1120 characters from rpb2, and 20 collections and 1035 characters from tef1-α. The final dataset comprised 82 collections and 4233 characters from ITS+LSU+rpb2+tef1-α. After checking for unsupported conflicts (BS < 70%) between single-gene maximum likelihood (ML) phylogenies, a concatenated four-locus dataset was assembled. Phylogenetic analysis using ML was performed, followed by manual adjustments in raxmlGUI 2.0.13, along with Bayesian analysis, both conducted on the CIPRES Science Gateway version 3.3 web server (Miller et al. 2010), available at https://www.phylo.org/. A mixed-model (partitioned) scheme was employed for 72 MycoKeys 123: 69–88 (2025), DOI: 10.3897/mycokeys.123.160438 Didsanutda Gonkhom et al.: Three new Scleroderma species and a record from northern Thailand both ML and Bayesian analyses, with the alignment split into four-character sets: ITS, LSU, rpb2, and tef1-α. The best-fit substitution models of jModelTest2 version 2.1.6 (Darriba et al. 2012) in XSEDE were chosen for Bayesian analysis. The selected models were HKY+G for ITS, GTR+I+G for nrLSU, HKY+G for rpb2, and SYM+I+G for tef1-α. Four independent runs, each with four chains, were performed for 1,000,000 generations, with sampling every 100 generations. The average standard deviation of split frequencies at the end of the runs was 0.015009. The burn-in phase (25%) was determined by assessing stationarity in the generation-likelihood plot using Tracer version 1.7.1 (Rambaut et al. 2018). The resulting phylogenetic tree was visualized in Treeview 32 and further edited using Adobe Illustrator CS6.0. Table 1. Phylogenetic analysis list of species, herbarium number, place of origin, and GenBank accession number. Species Voucher information Location GenBank accession no. Reference ITS LSU Rpb2Tef1-α Pisolithus aurantioscabrosus AWW297 Malaysia EU718112 EU718146 FJ536648 FJ536681 Wilson et al. (2011) Scleroderma areolatum AWW211 USA EU718115 EU718149 FJ536651 FJ536683 Wilson et al. (2011) S. areolatum PBM2208 Australia N/A EU718150 FJ536652 FJ536684 Wilson et al. (2011) S. areolatum TNS:F-82295 Japan OQ025272 OQ025269 N/A N/A Kasuya et al. (2023) S. areolatum Kasuya-B4422 Japan OQ025273 OQ025270 N/A N/A Kasuya et al. (2023) S. areolatum O3C_4 USA JX030282 N/A N/A N/A Bzdyk et al. (2018) S. areolatum 23 Spain MN684210 N/A N/A N/A - S. areolatum Db-K -MH040288 N/A N/A N/A Bzdyk et al. (2018) S. areolatum Bk-N -MH040301 N/A N/A N/A Bzdyk et al. (2018) S. bermudense BZ3961 Belize EU718118 DQ644137 FJ536654 FJ536686 Wilson et al. (2011) S. bermudense EUA09 -OQ351725 N/A N/A N/A Bullaín-Galardis et al. (2024) S. bermudense SUA03 -OQ351729 N/A N/A N/A Wilson et al. (2011) S. bovista MCA242 USA EU718117 DQ644138 FJ536653 FJ536685 Wilson et al. (2011) S. citrinum AWW212 USA EU718119 EU718151 FJ536655 FJ536687 Wilson et al. (2011) S. citrinum F-PRL5772 USA GQ166907 N/A N/A N/A Zhang et al. (2013) S. citrinum K (M) 17485 England EU784413 N/A N/A N/A Zhang et al. (2013) S. citrinum CITSCL1 USA FM213344 N/A N/A N/A Zhang et al. (2013) S. citrinum K (M) 53906 England EU784414 N/A N/A N/A Zhang et al. (2013) S. columnare CUB:Microbiology KHS3 Thailand AB459512 N/A N/A N/A Ruankaew Disyatat et al. (2016) S. columnare Scl1 Thailand AB854700 N/A N/A N/A Kaewgrajang et al. (2023) S. columnare CUB:Microbiology KHS10 Thailand AB459519 N/A N/A N/A Ruankaew Disyatat et al. (2016) S. columnare MFLU25-0110 (DG150) Thailand N/A N/A PX137624 PX126632 This study S. columnare MFLU25-0111 (DG153) Thailand PV444716 N/A PX137625 PX126633 This study S. dictyosporum IR250 Burkina Faso FJ840444 N/A N/A N/A Sanon et al. (2009) S. dictyosporum IR408 Burkina Faso FJ840445 N/A N/A N/A Sanon et al. (2009) S. meridionale AWW218 USA EU718121 EU718152 FJ536656 FJ536688 Wilson et al. (2011) S. mcalpinei OSC 24605 -EU718122 DQ682999 FJ536657 N/A Wilson et al. (2011) S. nitidum UFRN:Fungos 2034 Brazil KU759904 KU759903 N/A N/A Raut et al. (2020) S. nitidum UFRN:Fungos 2219 Brazil KU759908 N/A N/A N/A Raut et al. (2020) S. polyrhizum AWW216 USA EU718123 EU718153 FJ536658 FJ536689 Wilson et al. (2011) S. polyrhizum MA:Fungi-39352 Spain MT270662 N/A N/A N/A Ortiz-Rivero et al. (2021) S. separatum Ge5394 China OQ554975 N/A N/A N/A Wu et al. (2023) S. separatum ZLR31 China OQ554974 N/A N/A N/A Wu et al. (2023) S. separatum Ge4148 China OQ554973 N/A N/A N/A Wu et al. (2023) S. separatum MFLU 19-1347 (NTF066) Thailand PV444715 PV446742 N/A N/A This study 73 MycoKeys 123: 69–88 (2025), DOI: 10.3897/mycokeys.123.160438 Didsanutda Gonkhom et al.: Three new Scleroderma species and a record from northern Thailand Species Voucher information Location GenBank accession no. Reference ITS LSU Rpb2Tef1-α S. sinnamariense SINSCL3 (SCLN) Thailand FM213358 N/A N/A N/A Phosri et al. (2009) S. sinnamariense 150728-29 China MH513635 N/A N/A N/A Zhang et al. (2020) S. sinnamariense SINSCL1 (SCLK4) Thailand FM213356 N/A N/A N/A Phosri et al. (2009) S. sinnamariense SINSCL6 (SCLD1) Thailand FM213361 N/A N/A N/A Phosri et al. (2009) S. sinnamariense SINSCL4 (SCLY5) Thailand FM213359 N/A N/A N/A Phosri et al. (2009) S. sinnamariense CMU53:210-2 Thailand HQ687222 N/A N/A N/A Kumla et al. (2014) S. sinnamariense rpr-355 -MW374160 N/A N/A N/A Wang et al. (2022) S. sinnamariense HKAS122471 China ON794312 N/A N/A N/A Wang et al. (2022) S. sinnamariense SINSCL5 (SC1) Thailand FM213360 N/A N/A N/A Phosri et al. (2009) S. sinnamariense DX2021-8-2 -OL351633 N/A N/A N/A - S. sinnamariense MFLU25-0112 (DG157) Thailand N/A N/A PX137633 PX207694 This study S. sinnamariense MFLU25-0113 (DG158) Thailand PV444717 N/A PX137632 PX207695 This study S. sinnamariense MFLU25-0114 (DG159) Thailand PV444718 N/A PX137631 PX137634 This study S. sinnamariense MFLU25-0115 (DG160) Thailand PV444719 N/A PX137630 PX126634 This study S. sinnamariense MFLU 19-1647 (MO-DG020) Thailand PV444720 N/A PX137626 PX126635 This study S. sinnamariense MFLU 19-1648 (MO-DG021) Thailand PV444721 N/A N/A N/A This study S. sinnamariense MFLU 19-1649 (MO-DG022) Thailand PV444722 PV446743 PX137627 PX126636 This study S. sinnamariense MFLU 19-1650 (MO-DG023) Thailand PV444723 N/A PX137628 N/A This study S. sinnamariense MFLU 19-1652 (MO-DG034) Thailand PV444724 N/A N/A N/A This study S. sinnamariense MFLU 19-1653 (MO-DG035) Thailand PV444725 N/A PX137629 N/A This study S. sinnamariense MFLU 19-1341 (NTF012) Thailand PV444726 N/A N/A N/A This study Scleroderma sp. AWW260 Malaysia EU718124 EU718155 FJ536660 FJ536691 Wilson et al. (2011) Scleroderma sp. AB96 Cameroon KR819100 N/A N/A N/A Michaëlla Ebenye et al. (2017) Scleroderma sp. YAAS-L5455 -MT876542 N/A N/A N/A - Scleroderma sp. YAAS-L5449 -MT876541 N/A N/A N/A - Scleroderma sp. SL2085 Singapore OR354966 N/A N/A N/A - Scleroderma sp. ECM26-SERS -DQ146385 N/A N/A N/A Yuwa-Amornpitak et al. (2006) Scleroderma sp. LH35 Malaysia GQ268582 N/A N/A N/A Peay et al. (2010) Scleroderma sp. MFLU 19-1348 (NTF090) Thailand PV444728 N/A N/A N/A This study Scleroderma sp. MFLU 19-1517 (DMSLDG005) Thailand PV444729 N/A N/A PV749898 This study S. suthepense AWW311 Malaysia EU718125 EU718156 FJ536661 FJ536692 Wilson et al. (2011) S. suthepense CMU:55-SC2 Thailand NR_132871 N/A N/A N/A Kumla et al. (2013) S. suthepense JH-2016-0727-052 China MH513626 N/A N/A N/A Zhang et al. (2020) S. suthepense 180508-08 China MH513625 N/A N/A N/A Zhang et al. (2020) S. suthepense MFLU25-0109 (DG146) Thailand N/A N/A N/A PX126630 This study S. suthepense MFLU 19-1344 (NTF053) Thailand PV444727 N/A N/A N/A This study S. xanthochroum AWW254 Malaysia EU718126 EU718154 N/A N/A Wilson et al. (2011) S. yunnanense HKAS80386 -MW493647 MW493703 N/A N/A Kasuya et al. (2023) S. yunnanense PERTH-7604645 China MT270651 N/A N/A N/A Ortiz-Rivero et al. (2021) S. yunnanense TNS:F-82294 Japan OQ025271 OQ025268 N/A N/A Kasuya et al. (2023) uncultured fungus ASV_419 -LR993736 N/A N/A N/A - uncultured fungus ASV_1014 -LR994331 N/A N/A N/A - Scleroderma longistipes MFLU 19-1655 (DG109) Thailand PV444712 PV446740 PX126607 PX121227 This study Scleroderma longistipes MFLU 19-1656 (DG110) Thailand PV444713 PV446741 PX121228 N/A This study Scleroderma magnisporum MFLU 19-1345 (NTF062) Thailand PV444714 N/A N/A N/A This study Scleroderma microcarpum MFLU 19-1349 (DG002) Thailand PV436898 N/A N/A N/A This study N/A: not available; species in this study are indicated in bold black. 74 MycoKeys 123: 69–88 (2025), DOI: 10.3897/mycokeys.123.160438 Didsanutda Gonkhom et al.: Three new Scleroderma species and a record from northern Thailand Results and discussion Based on Scleroderma species, they were formerly separated by morphology, with basidiome size and shape varying depending on soil and environment and basidiospore morphology (Watling 2006; Sanon et al. 2009; Kumla et al. 2013; Gonkhom et al. 2025). In this study, Scleroderma sp. nov., described from macromorphological and micromorphological traits together with phylogenetic analysis of ITS, LSU, rpb2, and tef1-α genes, includes three new species—Scleroderma longistipes, Scleroderma magnisporum, and Scleroderma microcarpum—from northern Thailand. This work enhances our understanding of the diversity of Scleroderma species. Macroscopically, the size, color, and type of dehiscence of the basidiome, as well as the color and thickness of the peridium, are crucial traits for identifying Scleroderma. Microscopically, the size, shape, and ornamentation of the basidiospores are employed to differentiate species of this genus. The basidiomes and basidiospores are similar in size and character to those of S. separatum, S. dictyosporum, and S. hypogaeum (Sims et al. 1995; Cortez et al. 2011; Guzmán et al. 2013). Our ITS, LSU, rpb2, and tef1-α sequence analyses clearly separate these species from other reticulate-spored Scleroderma species in the section. Phylogenetic analysis The combined dataset of four genes comprised 4233 bp (including gaps): 676 bp for ITS, 1402 bp for LSU, 1120 bp for rpb2, and 1035 bp for tef1-α. The best RAxML phylogram, with a final likelihood value of -22414.937168, is presented. The matrix had 1563 distinct alignment patterns with 68.28% undetermined characters or gaps. The estimated base frequencies were: A = 0.233328, C = 0.255317, G = 0.274489, and T = 0.236866. Substitution rates were AC = 1.330307, AG = 4.855565, AT = 1.388503, CG = 1.501262, CT = 8.964382, and GT = 1.000000. The gamma distribution shape parameter was α = 0.929050. The phylogram topology derived from Bayesian analysis was similar to that from ML analysis. Bootstrap values of ML ≥ 70% and Bayesian posterior probabilities (PP) ≥ 0.90 are indicated in Fig. 1. Phylogenetic trees inferred from ML and MrBayes analyses resulted in similar topologies; therefore, only the ML tree (ITS+LSU+rpb2+tef1-α) is shown, with both ML bootstrap (BS) values and Bayesian posterior probabilities (PP). In the phylogram, Scleroderma longistipes (MFLU 19-1655 and MFLU 19-1655) was closely related to Scleroderma sp. (uncultured ectomycorrhiza ECM26 SERS, LH35 from Malaysia, and SL2085 from Singapore) with high statistical support: 100% BS/1.00 PP, 81% BS/0.93 PP, and 90% BS/1.00 PP, respectively. Scleroderma microcarpum (MFLU 19-1347) was closely related to the uncultured fungi ASV 1014 and ASV 419, with statistical support of 70% BS/0.94 PP. Scleroderma magnisporum (MFLU 19-1345) was closely related to Scleroderma sp. YAAS L5449 and YAAS L5455, with statistical support of 74% BS/0.97 PP. The species most closely related in the phylogenetic tree to Scleroderma microcarpum (MFLU 19-1347) was S. dictyosporum (Voucher IR250), with a genetic distance of 11.81% (65/570) between ITS sequences. Scleroderma magnisporum (MFLU 19-1345) and S. microcarpum (MFLU 19-1347) showed a genetic distance of 21.34% (95/445), supporting their distinction as separate species. This result is consistent with previous molecular phylogenetic studies 75 MycoKeys 123: 69–88 (2025), DOI: 10.3897/mycokeys.123.160438 Didsanutda Gonkhom et al.: Three new Scleroderma species and a record from northern Thailand Figure 1. Phylogenetic tree obtained from the maximum likelihood analysis of Scleroderma species. Maximum likelihood tree obtained from the alignment of ITS, LSU, rpb2, and tef1-α sequences. The bootstrap consensus tree was inferred from 1000 replicates. Pisolithus aurantioscabrosus was included as an outgroup. that strongly support the recognition of Scleroderma species as genetically discrete lineages (Phosri et al. 2009; Nouhra et al. 2012; Wu et al. 2023). Scleroderma species are found in temperate, tropical, and subtropical regions, which may be related to the higher diversity of Scleroderma or ectomycorrhizal fungi in these climatic zones (Jeffries 1999; Brundrett et al. 2005; Pradhan et al. 2011; Wu et al. 2023). In addition, 11 species of Scleroderma (S. areolatum, 76 MycoKeys 123: 69–88 (2025), DOI: 10.3897/mycokeys.123.160438 Didsanutda Gonkhom et al.: Three new Scleroderma species and a record from northern Thailand S. bovista, S. cepa, S. citrinum, S. dictyosporum, S. flavidum, S. lycoperdoides, S. polyrhizum, S. sinnamariense, S. verrucosum, and S. suthepense) have been recorded in Thailand based on morphology (Chandrasrikul et al. 2011; Kumla et al. 2013; Gonkhom et al. 2025). Phylogenetic analysis confirmed the placement of Scleroderma separatum based on the ITS and LSU regions (Fig. 1). S. separatum exhibits similar shapes and sizes of basidiomes and basidiospores (Wu et al. 2023). Phylogenetic analysis based on ITS and LSU sequences facilitated confirmation of the species we analyzed, which has been officially recorded in Thailand. Taxonomy Scleroderma longistipes Gonkhom, Sysouph. & Thongkl., sp. nov. Index Fungorum: IF903880 Fig. 2 Diagnosis. Epigeous brown to burnt umber basidiomata with long stipe, rubbery pale brown peridium, hyaline to yellow brown hyphae in exoperidium, hyaline hyphae in endoperidium, globose dark brown basidiospores with echinulate or spinose ornamentation. Holotype. Thailand • Chiang Rai Province, Mueang Chiang Rai District, Mae Fah Luang University campus, 04 June 2019, collected by Didsanutda Gonkhom, DG109 (MFLU 19-1655). Etymology. The species name (longistipes) refers to the long stipe of the basidiomata. Description. Basidiomata epigeous, 28–35 mm in diam., 42–60 mm high, club-shaped, with globular peridial head; with cracked to squamulose surface, brown (6E5) background when young, with fawn (7E4) to brown (6E5) or burnt umber (6F6) squamular cracks upon luteous background, hard skin, tough when mature. Stipe sub-cylindric, fat, with small irregular cracks at the top of the strip, 35–45 × 10–13 mm, white background, covered with brown (6E5) fibrillose squamules. Rhizomorphs more aggregated at the base, white, branched, and narrowing towards the base. Context white in peridium and stipe, turned dull red to greyish red (98B4-5) when cut. Peridium up to 5 mm wide when fresh, rubbery in consistence, pale brown (6D5). Peridium layer formed by simple-septate hyphae. Exoperdium slightly thickened walls, composed of interwoven to ramified and superimposed hyphae, hyaline to yellow brown, 2.9–3.8 µm diam. Endoperdium thick, composed of interwoven hyphae, hyaline, 4.3–7.2 µm diam. Clamp connections present on endoperidium hyphae. Gleba brownish grey (9E2), greyish brown (9E3), or oxblood red (9E7) to dark brown (9F4-7), compact, and powdery when mature. Basidiospores (n = 50) globose, echinulate, dark brown in KOH, (13.4–)14.5–17.2(19.5) µm in diam., with brown spinose ornamentation (2.6–5.9 µm high). Basidia not seen. Habitat and distribution. Caespitose or fasciculated on soil, epigeous, in northern Thailand. Additional specimens examined. Thailand • Chiang Rai Province, Mueang Chiang Rai District, Mae Fah Luang University campus, 04 June 2019, collected by Didsanutda Gonkhom, DG110 (MFLU 19-1656). Notes. Scleroderma longistipes is characterized by a larger brown basidiomata with a longer stipe that turns from dull red to greyish red when touched. The 77 MycoKeys 123: 69–88 (2025), DOI: 10.3897/mycokeys.123.160438 Didsanutda Gonkhom et al.: Three new Scleroderma species and a record from northern Thailand Figure 2. Scleroderma longistipes (MFLU 19-1655, holotype). A, B. Basidiomata; C. Scale on peridium surface; D. Cut side of peridium of MFLU 19-1655; E, F. Basidiomata; G. Scale on peridium surface; H. Cut side of peridium of Scleroderma longistipes (MFLU 19-1656); I. Exoperidial hyphae; J. Endoperidial hyphae; K. Clamped hyphae of endoperidium; L–Q. Basidiospore. Scale bars: 10 mm (A, B, E, F); 5 mm (C, D, G, H); 50 µm (I, J); 20 µm (K); 10 µm (L–Q). 84 MycoKeys 123: 69–88 (2025), DOI: 10.3897/mycokeys.123.160438 Didsanutda Gonkhom et al.: Three new Scleroderma species and a record from northern Thailand 3 Peridium yellowish to orangish-yellow, smooth to cracked ..............S. cepa – Peridium brown, with distinct warts or scales ............................................4 4 Peridium covered with tough raised warts, yellow-brown ..........S. citrinum – Peridium cracked, scaly, roughened, brown.................................. S. bovista 5 Peridium thin, leathery, yellowish white .......................................................6 – Peridium thick, with distinct cracks or subscaly .........................................8 6 Basidiospores 7–8.5 µm, dark brown, reticulate ................S. dictyosporum – Basidiospores larger than 8.5 µm, spiny ......................................................7 7 Basidiospores 8–12 µm, round, net-like ridges .....................S. verrucosum – Basidiospores 10–15 µm, slightly roughened texture ............S. columnare 8 Peridium smooth, brown, tough, thick (up to 5 mm) ......... S. magnisporum – Peridium cracked or scaly, background yellowish.......................................9 9 Basidiospores 8.24–13.33 µm, rhizomorphs pale brown .....S. microcarpum – Basidiospores 12.31–16.49 µm, spines up to 3.9 µm ............S. separatum 10 Stipe well-developed, more than 3 cm long ...............................................11 – Stipe short, less than 3 cm, or absent ........................................................13 11 Stipe sub-cylindric, cracked at the top, basidiospores 13.4–19.5 µm ......... ...................................................................................................S. longistipes – Stipe short or irregular, basidiospores smaller (<14 µm) .........................12 12 Basidiospores 7–12 µm, peridium star-shaped when split .......S. polyrhizum – Basidiospores 8–13 µm, peridium smooth to scaly ............. S. suthepense 13 Peridium golden yellow, apex rupturing at maturity ................... S. flavidum – Peridium brown to ochraceous, scaly, or roughened ................................14 14 Peridium leathery, verrucose, yellowish to lemon-yellow ......S. sinnamariense – Peridium brownish, smooth to scaly, spore mass dark brown .................15 15 Basidiome surface smooth, spore mass clearly olive ......S. lycoperdoides – Basidiome rough or scaly, spore mass brown to dark brown ..... S. areolatum Overall, this study significantly advances our understanding of the diversity and taxonomy of Scleroderma species in northern Thailand, describing three new species—Scleroderma longistipes, S. microcarpum, and S. magnisporum— and reporting a new record, S. separatum, from Thailand. The research integrates comprehensive morphological analyses with molecular phylogenetic methods, utilizing sequences from four loci (ITS, LSU, rpb2, and tef1-α) to confirm the distinctiveness of these taxa. The findings contribute to our knowledge of Scleroderma and establish a basis for future ecological and evolutionary research on ectomycorrhizal fungi. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Use of AI No use of AI was reported. 85 MycoKeys 123: 69–88 (2025), DOI: 10.3897/mycokeys.123.160438 Didsanutda Gonkhom et al.: Three new Scleroderma species and a record from northern Thailand Funding The authors appreciate the kind support provided by the Research and Researchers Industries (RRI) (grant no. PHD62I0018/2562) and Mae Fah Luang University. Author contributions Didsanutda Gonkhom: methodology, data curation, writing—original draft. Phongeun Sysouphanthong: investigation, conceptualization, writing. Marc Stadler, Naritsada Thongklang, and Kevin D. Hyde: writing—review and editing, supervision. 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