Introduction
Scleroderma Pers. (Sclerodermataceae, Boletales) is one of the most adaptable and widespread genera of ectomycorrhizal fungi [1]. This genus is associated with several trees or shrubs, such as species belonging to Abies, Betula, Coccoloba, Eucalyptus, Nothofagus, Pinus, Populus, and Quercus [2]. Morphologically, this fungus is characterized by a gasteroid basidiome with globose to subglobose basidiospores showing reticulate to echinulate ornamentation [3]. Several species of Scleroderma are employed as medicinal mushrooms or known to be poisonous [4,5]. The Index Fungorum lists a total of 131 Scleroderma species (http://www.indexfungorum.org, accessed on 18 Mar. 2022), and approximately 25 species are recognized morphologically.
Since 2012, we have conducted field trips to investigate the diversity of Scleroderma species from South Korea. As a result, we have discovered and described two new records within Sclerodermataceae (S. laeve and S. nastii). In this study, descriptions of Scleroderma species from Korea, including their morphological features and a phylogenetic analysis, are provided. In addition, this study is the first to review and identify specimens of Scleroderma stored in herbaria and collected in the field in Korea.
Materials and methods
Morphological observations
A total of 21 samples were collected during a mycological survey to investigate the diversity of mushrooms conducted from 2012-2021. The samples were examined for identification based on their macroscopic and microscopic characteristics in the present study. Dried materials were mounted in distilled water and 5% KOH using an Olympus BX53 microscope (Olympus Corporation, Tokyo, Japan) and Jenoptik ProgRes C14 Plus Camera (Jenoptik Corporation, Jena, Germany). Microscopic parameters were measured using ProgRes Capture Pro v.2.8.8. software (Jenoptik Corporation). A scanning electron microscopy (SEM) analysis of basidiospores was also performed. The systematics of the taxa included in this study were in accordance with the Index Fungorum (http://www.indexfungorum.org). Dried specimens were deposited in the herbarium of the Korea National Arboretum (Table 1).
Table 1. Scleroderma specimens used in this study, including specimen numbers, localities, internal transcribed spacer (ITS), and GenBank accession numbers.
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DNA extraction, sequencing and phylogenetic analysis
For phylogenetic analysis, genomic DNA was extracted from the specimens using a DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA, USA). The ITS (internal transcribed spacer) regions of the rDNA were amplified with the ITS1 and ITS4 primers as previously described [6]. The polymerase chain reaction amplicons were purified using a QIAquick Purification Kit (Qiagen Inc.) and directly sequenced using an ABI Prism 377 Automatic DNA Sequencer (Applied Biosystems, Foster City, CA, USA) with a BigDyeTM Cycle Sequencing Kit (version 3.1; Applied Biosystems). Sequences belonging to Sclerodermataceae were downloaded from the GenBank database (National Center for Biotechnology Information; https://www.ncbi.nlm.nih.gov/). These sequences and sequences of an outgroup taxon, Astraeus odoratus (GenBank Accession No. AJ629874), were used for analysis. The dataset was aligned using MAFFT v.7 [7]. All other parameters were set to the default values. A phylogenetic tree was constructed using RAxML in the CIPRES Science Gateway (https://www.phylo.org). The relative robustness of the individual branches was estimated by bootstrapping with 1,000 replicates.
Results
Phylogenetic analysis
A total of 21 ITS sequences were obtained from Scleroderma specimens in this study. As a result, the phylogenetic positions of five species (S. areolatum, S. bovista, S. citrinum, S. laeve, and S. nastii) were resolved by conducting an RaxML analysis of the ITS sequences (Fig. 1). They together formed well-supported clades. The genus Scleroderma is morphologically divided into three sections (Reticulatae, Scleroderma, and Sclerangium) based on the surface structure of basidiospores and the presence of clamp connections [2,8]. According to the present phylogenetic analysis, S. laeve belongs to S. sect. Reticulatae and S. nastii belongs to S. sect. Scleroderma. Our phylogenetic analysis showed that the taxonomic study was well supported by ITS sequences.
Taxonomy
Scleroderma laeve Lloyd, Mycol. Writ. (Cincinnati) 5 (Letter 63):11. 1916. (Figs. 1 and 2)
Korean name: Keun-Eorialbeoseot (큰어리알버섯); derived from the ‘big basidiome’ in Korean.
Description. Basidiome globose to subglobose, 15-45 mm diameter. Peridium thin, 1.0-1.5 mm thick, pale yellowish-brown, finally reddish-brown smooth to warty due to irregular cracking. Gleba compact, dark grayish-brown or reddish-brown. Basidiospores n=25, globose to subglobose, 10.0-13.0 µm in diameter including ornaments or 9.0-12.5 µm in diameter excluding ornaments; average=11 µm. Basidia not observed. Clamp connections absent.
Habitat. Growing on soil or sand, July to September.
Specimens examined. Korea. Jeollanam-do, Suncheon, Jul. 15, 2014, KA14-0542; Gyeonggi-do, Pocheon-si, Sept. 4, 2014, KA14-1374; Gyeonggi-do, Pocheon-si (37°44ʹ57.67ʺN 127°9ʹ59.44ʺE, alt. 112 m), Jul. 28, 2016, KA16-0827; Gyeonggi-do, Goyang-si (37°39ʹ32.98ʺN 126°45ʹ38.32ʺE, alt. 16.8 m), Sept. 18, 2018, KA18-1018.
Remarks. Based on the similarity of its morphological characteristics (shapes and size of the basidiome) to reported descriptions, this species was considered to be S. areolatum in Korea. However, the rhizomorph of S. laeve is well developed, and S. areolatum has a sessile or short pseudostipitate rhizomorph. (Fig. 2) [9]. The description of S. laeve collected from Korea was similar to Guzmán’s description [10]. According to a previous report by Kasuya et al. [11], this species occurs together with Lithocarpus edulis (Fagaceae) and sandy soil. The Korean samples were usually collected on soil, mainly in parks and gardens to date.
Scleroderma nastii Raut, in Raut, Basukala, Shrestha & Poudel, Stud. Fung. 5(1):53. 2020. (Figs. 1 and 3)
Korean name: Jageun-Eorialbeoseot (작은어리알버섯); derived from the ‘small basidiome’ in Korean.
Description. Basidiome epigeous, globose to subglobose, 10-15 to 10-20 mm, smooth, somewhat areolated, blackish-brown. Peridium 1 mm or less than 1 mm thick, pseudostipitate. Gleba compact, fleshy to dusty, blackish-brown. Basidiospores n=25, globose to subglobose, thick-walled, subreticulated to irregularly reticulated, 8.0-11.0 µm in diameter including ornaments or 7.5-10.0 µm in diameter excluding ornaments; average=9 µm. Basidia not observed. Clamp connections absent.
Habitat. Solitary to caespitose on soil under leaves and litter of Pinus species.
Specimens examined. Korea. Gyeonggi-do, Pocheon-si, under Pinus koraiensis, Jul. 12, 2012, KA12-0553; Gyeonggi-do, Pocheon-si, under P. koraiensis, Jul. 27, 2012, KA12-0905; Gyeongsangnam-do, Sancheong-gun, 20 Aug. 2013, KA13-0718; Gyeonggi-do, Pocheon-si, under mixed forest, Oct. 30, 2013, KA13-1555; Gyeonggi-do, Pocheon-si (37°45ʹ12.59ʺN 127°9ʹ55.52ʺE, alt. 109 m), Jul. 28, 2016, KA16-0813.
Remarks. According to the original description of S. nastii [12], the basidiospores were described as showing an irregular reticulum under SEM. However, SEM photos of the specimens in Korean collections showed that this species had somewhat echinulate basidiospores (Fig. 3). In addition, an original description [12] showed that the habitat of the species was the leaves and litter of Quercus trees. However, the Korean samples were often collected from the litter of Pinus trees or mixed forests. These findings indicate that this species occurs not only on Quercus but also on Pinus. This is the second record of the species since the original description.
Discussion
In Korea, seven species of Scleroderma (S. areolatum, S. bovista, S. cepa, S. citrinum, S. flavidum, S. lycoperdoides, S. verrucosum) have been recorded (Korea National Arboretum; ) [13]. In the phylogenetic analysis, the phylogenetic positions of five species (S. areolatum, S. bovista, S. citrinum, S. laeve, and S. nastii) were confirmed based on ITS regions (Fig. 1) [14]. The species of genus Scleroderma show similar shapes and sizes of their basidiomes, but phylogenetic analysis based on ITS sequences allowed the Scleroderma species that we analyzed to be easily identified. Furthermore, S. areolatum can be recognized as the most common species in Korea. Although four Scleroderma species (S. cepa, S. flavidum, S. lycoperdoides, and S. verrucosum) have been officially recorded in Korea, these species have not been collected in our field forays since 2012. Among these species, S. verrucosum has been recorded as a type species in the genus Scleroderma (index Fungorum; http://www.indexfungorum.org/). Therefore, additional collection efforts are needed to confirm their existence in Korea. Based on the specimens collected in this study, a morphological taxonomic key for five species from South Korea is provided below.
Key to Scleroderma species from South Korea
1. Basidiospores subreticulated or reticulated··········································································2
- Basidiospores echinulated ·····························································································3
2. Basidia not observed ·························································································S. bovista
- Basidia present ·······························································································S. citrinum
3. Rhizomorphs well developed ·························································································4
- Rhizomorphs sessile to shortly substipitate ···························································S. areolatum
4. Basidiospores small, > 10 µm ················································································S. nastii
- Basidiospores larger, < 10 µm ················································································S. laeve
