Gyromitra is a genus of Ascomycota, and its species commonly referred to false morels, exhibit a mycorrhizal association. Some Gyromitra species, including G. esculenta, G. fastigiata, G. gigas, and G. infula, cause severe poisoning and even death humans the presence of toxic gyromitrins . Gyromitra has a taxonomic history of transfers among various genera, including Discina, Helvella, and Maublancomyces. Traditional characters have been used to segregate species, including their ecological features and macro- and micro-scopic characteristics. . Five subgenera have been recognized in Gyromitra: Caroliniana, Discina, Gyromitra, Melaleucoides, and Pseudorhizina . Recently, the subgenus Pseudoverpa was described based on its unique morphoecological profile of the Cypriot collections . In addition, internal transcribed spacer (ITS) and Large subunit (LSU) phylogenetic analyses showed that collections from Cyprus in a well-supported clade within Gyromitra distant from their closest neighbor by 26 positions at the LSU locus.
To date, two species of Gyromitra (G. esculenta and G. infula) have been recorded in Korea . In this study, we analyzed the morphological characteristics and sequence data of three unrecorded Gyromitra species to confirm their taxonomic position.
MATERIALS AND METHODS
Fungal materials and morphological observation
Twelve specimens of Gyromitra have been collected during mushroom field foray since 2009. Macro-morphological characteristics were examined using a dissecting microscope. For characterization, the paraphyses, asci, and ascospores were mounted in distilled water and 5% KOH, examined using an Olympus BX53 compound microscope (Olympus, MA, United States), and photographed with a Jenoptik ProgRes C14 Plus camera (Jenoptik Corporation, Jena, Germany). Microscopic parameters were measured and calculated using ProgRes Capture Pro v. 2.8.8 software (Jenoptik Corporation). The dried specimens were deposited in the Korea National Arboretum (KH).
DNA isolation, ITS sequencing, and phylogenetic analysis
Genomic DNA was extracted from 12 specimens using the DNeasy Plant Mini DNA Extraction Kit (Qiagen Inc., Valencia, CA, USA), following the manufacturer’s instructions, and used for PCR amplification. ITS 1 and 2, along with 5.8S rDNA were amplified using the primers ITS1 and ITS4 . The 28S nuclear ribosomal LSU was amplified using the primers LR0R and LR6 . The sequences generated in this study were assembled using Chromas v. 2.6.6, and then edited using BioEdit v. 7.2.5  and MEGA v. 7.0.26 . equences were assembled and deposited in GenBank. The ITS and LSU accession numbers are in Table 1. For phylogenetic analyses, the were aligned using MAFFT v. 7.475  using the auto-option mode. Phylogenetic trees were constructed using RAxML in the CIPRES Science Gateway v.3.3 portal . The relative robustness of the individual branches was estimated by bootstrapping with 1,000 replicates.
The RAxML tree showed that specimens of G. gigas, G. aff. perlata, and G. tianshanensis formed well-resolved monophyletic clades with high bootstrap values (100, 100, and 90, respectively). In addition, the phylogenetic positions of G. infula and G. esculenta collected from Korea were confirmed. In a previous report , G. gigas was considered a species complex comprising six different species. In this study, G. tianshanensis clustered with G. xinjiangensis and G. infula, but differed from the sister group. A search showed that three specimens of G. perlata from Korea were 100% identical to those from France (MG846993) and China (MG871303). In the ITS and LSU phylogenetic trees constructed, sequences from the Korean specimens clustered with G. perlata.
Gyromitra gigas (Krombh.) Cooke, Mycogr., Vol. 1. Discom. London (No. 5): 191. 1878. (Figs. 1-3)
Korean name: Keunmagwigombo-beoseot (큰마귀곰보버섯); derived from the species epithet ‘gigas’ that means “giant” in Latin.
Description: Apothecia stipitate, irregularly lobed, inflated, 5–12 cm when fresh, 1.5–3.5 cm dia and 2–3 cm high when dry; hymenium dull yellowish brown, cinnamon, reddish brown, dark brown to blackish when dry, undulate-rugose, receptacle surface white, cream, grayish white, light brown to black, glabrous; stipe subcylindrical, enlarged at the base, typically fluted with broadly rounded ribs, white, light brown to black, glabrous to subpubescent, internally hollow, 2–3 × 0.3–1.3 cm. Excipulum of textura intricate, 40–55 μm thick, hyphae hyaline, 35 μm dia. Asci (n = 20) cylindrical, tapering at the base, 8-spored, 270–330 × 17–21 μm, ave. 290–320 × 18.5–20 μm. Paraphyses cylindrical, septate, hyaline, 7–11 μm long. Ascospores (n = 20) ellipsoidal to subfusoid, hyaline, smooth, biguttulate, non-apiculate, irregularly biseriate when young, uniseriate at maturity, 26–32 × 11.5–14 μm, ave. 28–30 × 12–13.5 μm.
Material examined: Korea, Pyeongchang-gun, 2021, leg. C. S. Kim, KA21-0153.
Notes: Gyromitra gigas regarded as a species complex morphologically similar taxa. Recently, this species was separated into six taxa G. gigas (Krombh.) Cooke, G. montana Harmaja, G. korfii (Raitv.) Harmaja, G. ticiniana Littini, G. pseudogigas X.C. Wang & W.Y. Zhuang, and G. khanspurensis Jabeen & Khalid, on phylogenetic analysis . Species within the G. gigas species complex are characterized by stipitate ascomata are saddle-shaped to irregularly lobed or cerebriform and wrinkled, and yellow-brown to brown reddish brown, ribbed to sulcate, white to yellow-brown stipe, ellipsoid to fusiform ascospores that are roughened to finely reticulate and uniguttulate or triguttulate with an inconspicuous to distinctive apiculus that is up to 4 μm long . In addition, G. gigas has been reported only Europe and Asia . In 2020, the G. gigas species complex was divided into six species (G. gigas, G. montana, G. korfii, G. ticiniana, G. pseudogigas, G. khanspurensis) based on phylogenetic analyses and morphology . In this study, the Korean specimens formerly identified as G. gigas formed a monophyletic clade with other sequences of G. gigas sensu stricto.
Gyromitra aff. perlata (Fr.) Harmaja, Karstenia 9: 11. 1969. (Figs. 1, 2, and 4)
Korean name: Neolbeunmagwigombo-beoseot (넓은마귀곰보버섯); derived from the species epithet ‘perlata’. Latin meaning perlatus (wide)
Description: Apothecia cup-shaped, up to 7 cm high when fresh, smooth, wavy to wrinkled, or bumpy. Upper surface brown to reddish brown, darkening to dark brown or blackish. Under surface whitish to pale grayish brown or yellowish. Asci ( n = 20) subcylindrical, tapering at the base, 8-spored, 160–210 × 12.5–15 μm, ave. 180–200 × 13–14.5 μm. Paraphyses filiform, septate, orangish to orange-brown, 6–9 μm wide at apex. Ascospores (n = 20) fusoid, hyaline, smooth, apiculi pointed, 28–45 × 11–16 μm, ave. 32–42 × 13–15 μm.
Material examined: Korea, Pocheon-si, 2015, leg. S. K. Han, KA15-0013.
Notes: Based on the neotype specimen of G. perlata from France (MG846993), ascospores of this species were 40–46 × 13–17 μm. However, the ascospores of Korean G. perlata were 22.5–27 × 11–13 μm. Therefore, the size range of ascospores are quite different between French and Korean specimens . In a previous report , G. perlata was classified as a species complex. However, the Korean specimens of G. perlata cluster with the neotype of G. perlata from France (MG846993) with strong support. Therefore, this species identified as G. aff. perlata.
Gyromitra tianshanensis X.C. Wang & W.Y. Zhuang, Mycologia 111(1): 72. 2018. (Figs. 1, 2 and 5)
Korean name: Cheonsanmagwigombo-beoseot (천산마귀곰보버섯); derived from the species epithet ‘tianshanensis’. Mandarin tianshan (celestial mountains).
Description: Apothecia stipitate, cerebriform, irregularly lobed, convex, margin appressed and fused to the stipe, up to 4–6 cm high when fresh, 1.5–3.5 cm diameter and 2–3 cm high when dry; hymenium dull yellowish brown, cinnamon, reddish brown, dark brown to blackish when dry, undulate-rugose, receptacle surface white, cream, grayish white, light brown to black, glabrous; stipe subcylindrical, enlarged at base, typically fluted with broadly rounded ribs, white, light brown to black, glabrous to subpubescent, internally hollow, 2–3 × 0.3–1.3 cm. Excipulum of textura intricate, 100–220 μm thick, hyphae hyaline, 2–4 μm diameter. Asci (n = 20) subcylindrical, tapering at the base, 8-spored, 160–210 × 12–15 μm, ave. 170–200 × 13–14.5 μm. Paraphyses filiform, septate, hyaline, 7–9 μm wide at apex and 4.5–6 μm below. Ascospores (n = 20) narrowly ellipsoidal to fusoid, hyaline, smooth, biguttulate, non-apiculate, irregularly biseriate when young, uniseriate at maturity, 17–21.5 × 6–9.5 μm, ave. 18–21 × 7–9 μm.
Material examined: Korea, Ulleung-gun, 26 Sep. 2013, leg. S. K. Han, KA13-1244.
Notes: Gyromitra tianshanensis clusters with G. infula and G. xinjiangensis but in morphological and molecular sequence. G. tianshanensis was introduced as a new species in Xinjiang, China and was collected rotten wood at 1700 m . This species was collected from the rotten wood of Picea species at 1,900 m Ulleungdo Island, Korea. In China, Gyromitra species are usually associated with coniferous trees or grow in coniferous-deciduous mixed forests with Picea, Pinus, and Betula at altitudes of 800-4000 m . Therefore, Gyromitra species can be related to altitude and geographical position, especially G. tianshanensis. The Korean collection was the first record outside its type locality.