First record of Inosperma subsphaerosporum (Inocybaceae) in Indonesia

принадлежность наших образцов к кладе I. subsphaerosporum из типового материала. Настоящее сообщение представляет информацию о первой находке I. subsphaerosporum в Индонезии. Для анализа содержания пита-тельных веществ и мускарина в этой индонезийской

Inosperma subsphaerosporum, a new species of Inosperma was recently reported from Hainan Tropical Rainforest National Park China (Deng et al., 2021).The specific epithet "subsphaerosporum" refers to the shape of its basidiospores.Morphologi-cally, this species can be determined by the brown to yellowish, scaly-fibrillose cap, grayish to brownish dense lamellae, a cylindrical longitudinally fibrillose stipe with recurved fibrils at the apex, and a salty or musty smell (Deng et al., 2021).To date, the distribution of this species was only known from the type locality in Hainan Province (China), grow on soil and associated with Fagaceae (Deng et al., 2021).Previously, I. subsphaerosporum was phylogenetically placed in an Old-World tropical clade, in which all members are described from South Asia.However, knowledge of this species in Southeast Asia has yet to be found.
The information on wild Indonesian inocyboid mushroom is scarce, and species of Inosperma are poorly known.No information provided regarding the I. subsphaerosporum strain history, nor herbarium collection number that deposited in Indonesia.In 2022 and 2023, our field examination on the distribution of Inocybaceae in Central Kalimantan (Borneo) in Indonesia has revealed the consumption of wild inocyboid mushroom by the local community.The foraging and consuming of wild inocyboid mushrooms have been done for decades.During the fungus foray held by the Indonesian mushroom hunter community (KPJI), we obtained some basidiomata of wild edible inocyboid mushroom.The goal of this study was aimed to reveal the taxonomical position of our inocyboid specimens based on morphological and molecular evidence.

Material and Methods
The specimens were obtained at Buntok, Dusun Selatan, Barito Selatan, Central Kalimantan, Indonesia (1°42'30.3"S,114°49'25.8"E), in 2022 and 2023 during a mushroom hunting carried by the KPJI.The fruiting bodies were photographed in situ and ecological information (coordinate, substrate, vegetations) were noted.Some of the specimens were deposited to Herbarium Bandungense Indonesia with the collection number FIPIA-DEP59.The morphological characteristics were examined from fresh basidiomata in situ and in Mycology Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, IPB University, Indonesia and in the Integrated Laboratory of Bioproducts (iLaB), BRIN, Bogor, Indonesia.The macromorphological features including color, size, pileus and stipe ornamentation, margin, and lamellae were observed directly and by a loupe.The micromorphological characters of basidium, cystidia, spores (shape, size, color, ornamentation), and clamp connection were observed using light microscope.Specimens were also subjected to examination via scanning electron microscopy (SEM), which was prepared following the methods of Goldstein et al. (1992).Lamellae were cut into small pieces (5 × 5 mm), pre-fixed in 2.5 % glutaraldehyde of a cacodylate buffer with a pH of 8.4 at 27 °C for two days.Next, they were pre-fixed in 2 % tannic acid for six hours and washed with four different cacodylate buffers.The samples were dehydrated in 50-100 % ethanol series, infiltrated with t-butanol twice for 10 minutes, and freeze dried.Freeze dried samples were mounted on an aluminum stub with double-sided carbon tape and coated with gold.Samples observed using the JSM IT 200 SEM system (JEOL, Tokyo, Japan).The specimens were identified using related identification references (Matheny et al., 2019;Deng et al., 2021;Li et al., 2022).
Fresh basidiomata were used to isolate DNA.DNA was extracted using a Qiagen DNeasy Plant Mini Kit according to the manufacturer's instructions.A Thermo Scientific Arktik Thermal Cycler was used for DNA amplification (ThermoFisher-Scientific). The amplification reaction was carried out using the primer pairs ITS 5 (5'-GGA AGT AAA AGT CGT AAC AAG G-3') and ITS 4 (5'-TCC TCC GCT TAT TGA TAT GC-3') (White et al., 1990).The PCR amplification was performed in 50 µL total reaction containing 9 µL ddH2O, 1.5 µL of 10 pmol of each primer, 25 µL PCR mix from 2× PCR buffer for KOD FX Neo (Toyobo), and 2 µL 100 ng template DNA.The PCR condition was set as follows: initial denaturation at 94 °C for 5 minutes, followed by 30 cycles of denaturation at 94 °C for 30 seconds, annealing at 56 °C for 45 seconds, and extension at 72 °C for 1 minute.The final extension was set at 72 °C for 10 minutes.PCR product was analyzed using 1.5 % agarose gel electrophoresis run with TAE buffer (40 mm Tris-acetate, pH 8.0 1 mM EDTA), stained with FloroSafe DNA stain and visualized by the Gel Doc EZ Gel Documentation System (Biorad).PCR products were sent to the 1 st Base Malaysia for sequencing.
The sequences were assembled using Chro-masPro software.The final aligned sequences were deposited in GenBank (https://www.ncbi.nlm.nih.gov/) to obtain the accession number.The sequence was subjected to Basic Local Alignment Search Tool (BLAST) in NCBI to compare the homology with previous data.Selected published sequences based on BLAST results (Table ) were used for phylogenetic tree analyses with Auritella hispida as the outgroup.The phylogenetic tree of Randomized Axelerated Maximum Likelihood (RaxML) Black Box was generated on CIPRES (Stamatakis, 2014).All trees were then edited using TreeGraph Software version 2.9.2-622 beta.The Bootstrap value (BS) ≥ 70 % was shown on the branch of the phylogenetic tree.Fruiting bodies medium in size.Cap 4-7 mm, conical in young basidiomata, plano-convex to applanate in mature stages with an obtuse umbo, margin incurved at first and then slightly decurved in time, surface moist to dry, pileus radially fibrillose to rimulose with patches, scales of veil remnants, margin rough to split in in time, yellowish brown to brown around umbo, yellowish white with paler col-or compared to the central region towards the margin, golden yellow in very mature stages.Lamellae adnexed to sinuate, dense, with series of lamellula, 20-40 mm length, brownish to white, edge slightly serrate.Stipe 40-100 × 8-11 mm, cylindrical, hollow, equal from apex to base with a slightly swollen base, squamulose at the apex, fibrillose downwards, white to cream.Context solid, fleshy, white to cream.Basidiospores subglobose to globose, 5-7× 4-6 µm, smooth, yellowish to tan, with a prominent lipid body and an indentation in the center region when observed using SEM.Basidia clavate, notable oil inclusion, 26-31 × 7-8 µm in dimension, hyaline, 4-spored, sterigmata 3-4.5 µm in length.Pleurocystidia absent.Cheilocystidia abundant, 21-41 × 8-11 µm, in clusters, mostly club-shaped, septate, primarily thin-walled and some with thick-walled, hyaline, with yellow intracellular cytoplasm.Hyphal trama composed of cylindric to inflated hyphae, 12-15 µm in diameter, hyaline.Pileipellis is a cutis, golden yellow, with thin-walled, cylindrical hyphae, 7-10 μm in diameter.Oleiferous hyphae prominent, 3-4 μm wide, pale to yellow brown, smooth, occasionally dichotomously branched.Hymenial and stipe trama with dense interwoven hyphae, thin-walled, yellowish, 5-10 μm wide.Clamp connections are present in all tissues.
Habitat: gregarious near surrounding Euphorbiaceae and Arecaceae trees.
Distribution: To date, known only from Hainan Province, China.

Molecular Analyses
The aligned nucleotide sequence was deposited to GenBank with the accession number ITS OP880257.
The BLAST result revealed that our specimens had high similarity to Inosperma subsphaerosporum from type material (96 %) as the top hits.The phylogenetic tree (Fig. 6) constructed from ITS sequences resolved specimen FIPIA-DEP59 was in the same clade of I. subsphaerosporum from type material with 93 % BS value.The phylogenetic tree displayed that I. subsphaerosporum FIPIA-DEP59 was a sister clade to I. flavobrunneum.Inosperma subsphaerosporum FIPIA-DEP59 was in the different clade to morphologically similar species I. carnosibulbosum, I. akirnum, and I. saragum.First record of Inosperma subsphaerosporum (Inocybaceae) in Indonesia

Discussion
Currently, there are only eighty taxa of Inosperma (Inocybaceae) documented worldwide (Index Fungorum, 2023).Inosperma sphaerobulbosum was recently introduced as a new species from China by Deng et al. (2021).The current study reports for the first time the distribution of this species aside from the type locality (Hainan Province, China) and contributes a new information to Indonesian macrofungi.Our work revealed the extension of geographical distribution of I. sphaerobulbosum in Kalimantan (Indonesia), which approximately 2474 km away from the type locality.Previously, Putra and Hafazallah (2020) reported the unidentified edible mushroom from Central Java (Indonesia) with the local name "jamur lengkuas" morphologically similar to I. sphaerobulbosum FIPIA-DEP59 which therefore warrants a further examination.In the sampling site, our specimens were collected from soil mixed with leaves litter.Larsson et al. (2009) emphasized that many Inocybe species in the section Rimosae (currently genus Inosperma) are known to occur on more nutrient rich soil.In addition, I. sphaerobulbosum FIPIA-DEP59 grows near Euphorbiaceae and Arecaceae which dominate the surrounding area and in contrast to Deng et al. (2021) reported the specimens were collected near fagaceous trees.The Inocybaceae is known as family of ectomycorrhizal mushroom-forming fungi (Matheny et al., 2019), however the host plant of I. sphaerobulbosum FIPIA-DEP59 remains unclear.
In the field, I. sphaerobulbosum FIPIA-DEP59 can be recognized by its gregarious habit, mediumsized basidioma, having scaly-fibrillose pileus, and a fibrillose stipe.Inosperma sphaerobulbosum FIPIA-DEP59 has approximately similar of pileus dimension to I. carnosibulbosum (Pradeep et al., 2016) but larger in size when compared to I. akirnum, and I. saragum Latha and Manimohan (2017).Some ba- sidiomata of I. sphaerobulbosum FIPIA-DEP59 displays pileus with layer of universal veil.Deng et al. (2021) emphasized that this feature is non persistent character and is influenced easily by the weather.The lamellar edge of I. sphaerobulbosum FIPIA-DEP59 was fimbricate and slightly serrate which in contrast to the related morphological similar species in having only fimbricate edge.Microscopically, the outline of basidiospores of I. sphaerobulbosum FIPIA-DEP59 differs it to the closely morphological species of I. carnosibulbosum, I. akirnum, and I. saragum.The spore dimension of our specimen was slightly lower than the three closely related species.However, in line with those species, I. sphaerobulbosum FIPIA-DEP59 has abundant cheilocystidia in clusters.In addition, the yellow intracellular contents of cheilocystidia distinguished our specimen from the genus Pseudosperma.
A comparison of the ITS sequence of specimen FIPIA-DEP59 derived from Central Kalimantan with the available taxa information from GenBank indicated that BLAST queries led to I. sphaerobulbosum from type material as the top hits.Accordingly with BLAST result, the phylogenetic tree committed that our Inosperma was identified as I. sphaerobulbosum.The phylogenetic placement of our specimen in the same clade as I. sphaerobulbosum from Hainan, China with 93 % BS value.In the phylogenetic tree, the position of clade of I. sphaerobulbosum FIPIA-DEP59 was mixed between Old World tropical clade 1 and Old World tropical clade 2 (Pradeep et al., 2016;Latha, Manimohan, 2017;Matheny et al., 2019;Aïgnon et al., 2021) due to only single gene used in this study.Previous study confirmed that Old World tropical clade 2 was composed of Inosperma species from India, Thailand, Papua New Guinea, and China (Matheny et al., 2019;Deng et al., 2021).Inosperma sphaerobulbosum is reported for the first time for Indonesia and enriches the knowledge of the distribution of Old World tropical clade 2. Our study provides the available morphological and molecular information of I. sphaerobulbosum in Indonesia.In the research site, I. sphaerobulbosum is considered as delicacy mushroom and traded at local market as well as by using the social media.The mushroom is prized Rp. 18000/kg or US$1.15/kg.
The indigenous people usually forage this species around September to December, and sometimes March to April when the rain and relative humidity are high.Considering the edibility of this species, we suggest that the nutritional analysis and cultivation efforts of I. sphaerobulbosum needs a warrant in Indonesia, as many wild mushrooms are recognized as source of secondary metabolite and medicinal properties (Bukharova et al., 2020).However, many species of Inosperma were reported to cause neurotoxic poisoning in humans after consumption worldwide (Bijeesh et al., 2020;Parnmen et al., 2021;Deng et al., 2022;Li et al., 2022).For example, I. erubescens caused mass poisoning in Germany (Lurie et al., 2009), 10 cases of poisoning caused by Inosperma species in Thailand from 2010 to 2018 (Parnmen et al., 2021), and recently I. zonativeliferum caused poisoning in 10 people in China (Deng et al., 2022).Li et al. (2022) reported that I. sphaerobulbosum contains none of the tested neurotoxins.Although no report indicates the poisoning case by consumption of this mushroom from type locality as well as in our sampling site, we expect to determine the four main mushroom neurotoxins (muscarine, psilocybin, ibotenic acid, and muscimol) contents of I. sphaerobulbosum FIPIA-DEP59 and closely related species in the near future.

Conclusion
The current study reports the first distribution of Inosperma subsphaerosporum in Indonesia and new information aside from type locality (China).
Morphologically, I. sphaerobulbosum FIPIA-DEP59 can be determined by the conical applanate cap with umbo, incurved to decurved margin, dry pileus with fibrillose to rimulose surface, lamellae adnexed to sinuate, cylindrical stipe, subglobose to globose smooth basidiospores, and abundant cheilocystidia in clusters.The BLAST result and the phylogenetic tree confirmed our specimens as I. subsphaerosporum.Future study should be done to analyze both nutritional and mushroom neurotoxins content of this Indonesian Inosperma.

Table Species ,
outgroup, herbarium voucher, and GenBank accession numbers used in this study