Eight novel cave fungi in Thailand’s Satun Geopark

Abstract

Karst caves are unique oligotrophic ecosystems characterised by the scarcity of organic litter, darkness, low to moderate temperatures, and high humidity, supporting diverse fungal communities. Despite their importance, little is known about the fungi in karst caves in Thailand. In 2019, we explored the culturable mycobiota associated with three selected types of substrates (air, soil/sediment and organic litter samples) from two karst caves, the Le Stegodon and Phu Pha Phet Caves, in the Satun UNESCO Global Geopark in southern Thailand. Based on morphological characters and multilocus phylogenetic analyses, eight new species (Actinomortierella caverna, Hypoxylon phuphaphetense, Leptobacillium latisporum, Malbranchea phuphaphetensis, Scedosporium satunense, Sesquicillium cavernum, Thelonectria satunensis and Umbelopsis satunensis) were described, illustrated, and compared to closely related species. These new fungal taxa form independent lineages distinct from other previously described species and classified into eight different families across six orders and two phyla (Ascomycota and Mucoromycota). This paper provides additional evidence that the karst caves located within the Satun UNESCO Global Geopark, situated in the southern region of Thailand, harbour a diverse range of newly discovered species.

Citation: Preedanon S, Suetrong S, Srihom C, Somrithipol S, Kobmoo N, Saengkaewsuk S, Srikitikulchai P, Klaysuban A, Nuankaew S, Chuaseeharonnachai C, Chainuwong B, Muangsong C, Zhang ZF, Cai L, Boonyuen N (2023). Eight novel cave fungi in Thailand’s Satun Geopark. Fungal Systematics and Evolution 12: 1–30. doi: 10.3114/fuse.2023.12.01

INTRODUCTION

Fungi constitute a remarkably diverse group of organisms that exhibit a wide range of morphological, ecological, metabolic, and phylogenetic characteristics. They can be found in various forms, including single-celled and multicellular filamentous structures. Fungi play crucial roles in ecosystems as saprophytes, decomposers, mutualists, and pathogens (Schoch et al. 2009, Bastian et al. 2010, Naranjo-Ortiz & Gabaldón 2019). The current estimate of fungal diversity is highly uncertain, ranging from 2.2 to 3.8 M, but only approximately 146 000–150 000 species, or 3–4 %, are currently accepted taxa; thus, 96 % of fungal species remain unknown (Hawksworth & Lucking 2017, Banki et al. 2021). Fungal communities exist in every ecosystem on Earth, and the species compositions of the communities can be adjusted to adapt to various environmental conditions, including caves (Vanderwolf et al. 2013). Karst caves are dark, relatively cool, humid, nutrient-limited, and subterranean (Lee et al. 2012, Zhang et al. 2017, 2018). The unique environmental ecosystems in cave environments provide an opportunity to identify and study unusual fungi that are adapted to extreme conditions associated with oligotrophy (Gabriel & Northup 2013). According to research on karst cave fungi, they play important roles in the formation and characteristics of speleothems in caves by controlling major biogeochemical cycling and cave evolution (Vanderwolf et al. 2013, Zhu et al. 2022). Furthermore, it has been reported that the microbial communities in caves can obtain energy by breaking down aromatic compounds, fixing nitrogen from the atmosphere and interacting with minerals in terms of fungal metabolism and cave biogeochemistry (Boblitt et al. 2018, Jones & Northup 2021, Zada et al. 2021, Zhu et al. 2022). This evidence suggests that fungi living in extreme and harsh conditions might potentially produce novel compounds, bioactive secondary metabolites and/or enzymes that enable them to survive in these environments (Belyagoubi et al. 2018, Fernández-Remacha et al. 2022, Gubiani et al. 2022). Most of the fungal genera found in caves have also been isolated from natural substrates, such as sediment, cave walls, speleothems, guano, water, air, and various fauna (including bats) from different regions of the world; many of the fungi are not native to caves but have likely been introduced and dispersed by humans, fauna, water, and air currents (Jurado et al. 2008, Shapiro & Pringle 2010, Vanderwolf et al. 2013, 2016, Martin-Sanchez et al. 2014, Nováková et al. 2018, Carvalho et al. 2022, Leplat et al. 2022).

The cave-dwelling fungi found in the tropics, particularly in Thailand, are not well documented. In a recent study, Nuankaew et al. (2022) discovered two new species of Talaromyces in soil samples collected from the Satun UNESCO Global Geopark in southern Thailand. As part of a project that addresses the mycological diversity of the Satun UNESCO Global Geopark (Satun province, Thailand), two karst caves (Phu Pha Phet Cave and Le Stegodon Cave) were surveyed in this study. The fungal species in these two caves were identified using culturable morphotypes and BLAST queries against the fungal sequence database. Morphological characteristics, in combination with molecular data from seven loci (the ribosomal small subunit (SSU), the ribosomal large subunit (LSU), translation elongation factor 1-alpha (TEF-1α), beta-tubulin (TUB), α-actin (ACT), RNA polymerase II largest subunit (RPB1) and second largest subunit of ribosomal polymerase II (RPB2) regions) summarised in a multi-locus phylogeny, permitted the characterisation, description and illustration of eight new fungal species found in these caves.

MATERIALS AND METHODS

Cave information and sampling sites

Satun Province in southern Thailand, known as the “Land of Palaeozoic fossils”, was designated as the first UNESCO Global Geopark in the country on 17 April 2018 (Cheablam et al. 2021, Nantakat & Vorachart 2021). The Satun UNESCO Global Geopark covers four districts of Satun Province: Thungwa, Manang, La-Ngu, and Muang Satun. The region is renowned for its high diversity of fossil species from the Palaeozoic Era as well as for its diverse karst topography, which includes limestone, sandstone, mudstone, shale, and chert.

Phu Pha Phet Cave, also known as the “Diamond Mountain Cave”, is located within the Satun UNESCO Global Geopark at an elevation of 21 m (7°7’25”N 99°47’54”E) in Thungwa, Manang District (Fig. 1). It is the fourth largest cave in the world and the largest cave in Thailand, covering an area of more than 0.16 ha, and is approximately 536.65 m in length. The cave, which has a high ceiling and a unique atmosphere, contains sparkling stalactites and stalagmites resembling diamond flakes. More than 20 chambers are present within the cave, which can be accessed via a wooden bridge and is illuminated by installed lights. This cave is open to tourists and has therefore experienced some level of anthropogenic disturbance. However, certain areas of the cave are closed to the public to assure the protection of natural resources and promote sustainable tourism. As a result, the number of visitors to the cave is limited. Overall, the level of human disturbance in the Phu Pha Phet Cave can be considered moderate (Nantakat & Vorachart 2021; https://www.dmr.go.th/department-of-mineral-resources-thailand; accessed 26 May 2023). The depicted areas in Le Stegodon Cave indicate the sampling sites/zones: A (A1–A5), B (B1–B5), and C (C1–C5) (Fig. 2).

Fig. 1.

Map of Thailand and location of the two sampling sites Satun UNESCO Global Geopark in Satun Province in the southern part of Thailand.

Fig. 2.

Map of visited caves. Sampling sites/zones in Le Stegodon Cave and Phu Pha Phet Cave are shown, as are the locations of sampling sites/zones A (A1–A5), B (B1–B5) and C (C1–C5) in Phu Pha Phet Cave.

Le Stegodon Cave (Tham Le Stegodon; 107 m elevation; 7°7’35”N 99°59’49”E), located in the Thung Wa Subdistrict, Thung Wa District, is a sea cave with seawater intrusions on its western outlet (Fig. 1). It consists of three winding tunnels that converge within the mountain and have a total length of approximately three to four km. The tunnels are 10–20 m wide and 10–20 m high. The western outlet of Le Stegodon Cave is connected to a brackish stream that flows through a mangrove forest. The water level in the cave is influenced both by the stream and by the daily tides from the western outlet. While the cave is open to tourists, certain areas are closed to the public for the same reasons as in the case of Phu Pha Phet Cave. The level of human disturbance in some areas of the cave is considered low to moderate. Each of the two caves in this study was divided into three main zones for fungal sampling purposes (Duangkrayom et al. 2018, Nantakat & Vorachart 2021). Additionally, the positions of sampling sites/zones A (A1–A5), B (B1–B5), and C (C1–C5) within Phu Pha Phet Cave are also illustrated (Fig. 2).

Sample collection and fungal isolation

Sampling (Fig. 3) was performed in November 2019. The collection and isolation methods used were specific to the type of sample collected as follows: 1) Air samples (A) were collected using the Koch sedimentation method (Zhang et al. 2017). For each air sample, 3–5 Petri dishes containing 2 % potato dextrose agar (PDA, Difco) containing two antibiotics (50 μg/mL ampicillin and 50 μg/mL streptomycin) were exposed to the atmosphere in the cave for 15–20 min. The dishes were then sealed with Parafilm and placed in zip-lock plastic bags (Jiang et al. 2017). 2) Soil samples (S) of approximately 10–20 g was collected from the top 1–5 cm surface, placed in zip-lock bags, and stored at 4 °C in an ice box until it could be transferred to the laboratory for analysis. 3) Organic litter (O) consisting of guano, other animal dung, carcasses, and plant debris were collected in plastic zip-lock bags and kept at 4 °C in an ice box. All samples were transferred to the laboratory and kept at 4 °C in a refrigerator until used for further fungal isolation.

Fig. 3.

Samples (soil sediment, organic litter)

Isolation of cave fungi

Samples were processed according to the methods described by Zhang et al. (2017, 2021). One gram of S or O was added to 9 mL of sterile water in a sterile 15 mL centrifuge tube, and shaken by hand or mechanically for 10 min. The suspensions of S were diluted to 10^-1–10^-5 and 10^-2–10^-5 for O. Two hundred mL of suspension at each concentration were spread on PDA containing streptomycin (50 µg/mL) and ampicillin (50 µg/mL); three replicate plates were prepared for each dilution. All fungal plates were incubated at room temperature (23–25 °C) and examined at 24 h intervals for 2–3 wk. Single colonies were picked from the plates and transferred to PDA plates without antibiotics.

Morphological characterisation

The size, colour, shape, and arrangement of sporangiophores and conidiophores, sporangia and conidiogenous cells (i.e., phialides), spores, and conidia (n = 10–30) were observed after placing the material in a drop of lactophenol blue and examining it using a light microscope (OLYMPUS CX31; Olympus Corporation, Japan) at high magnification, following published procedures and techniques (Seifert et al. 2011, Zhang et al. 2017, Nuankaew et al. 2022, Watanabe 2002). Measurements were made of fresh cultures suspended in water, and the fungi were photographed using a Nomarski differential interference contrast microscope (OLYMPUS DP70). Species were determined using a morphological species concept, but DNA sequences were used to support the morphological data (Crous et al. 2021, 2022).

Candidate strains of potential new species were identified based on comparison of their Internal Transcribed Spacers (ITS) sequences with those of known genera and species using BLASTn tool; if the highest match was less than 97 %, the strain was considered a potential new species, according to the method outlined by Zhang et al. (2017). Candidate strains of potential new species were transferred to new plates containing PDA, malt extract agar (MEA), oatmeal agar (OA) or cornmeal agar (CMA) based on the given genera. The cultures were incubated at room temperature (23–25 °C) until morphological characterisation and molecular studies were performed. Colony characteristics and pigment production were examined after 14 d. Colony size was measured after 7 d for fast-growing strains, after 14 d for the slower strains, and after 8 wk for the most fastidious strains. The colour codes used in the fungal description follow the “Methuen Handbook of Colour” (Kornerup & Wanscher 1978). Fungal karst cave cultures were examined periodically for the development of reproductive structures according to the modified method of Réblová et al. (2016).

To maintain axenic cultures of fungi from karst caves, slant agar plates containing PDA were inoculated with small pieces of mycelium or with spore suspensions. Fungal strains were selected based on their morphotypes and stored at 4 °C for further study; the stored cultures were transferred to fresh medium every 6 mo. Fungal cultures of new species are maintained in the Thailand Bioresource Research Center (TBRC;https://www.tbrcnetwork.org, accessed 26 May 2023) and the National Biobank of Thailand (NBT; https://www.nationalbiobank.in.th; accessed 26 May 2023), and dry cultures were deposited at the BIOTEC Bangkok Fungarium (BBH; https://www.nationalbiobank.in.th/microbe-services; accessed 26 May 2023). The new taxa were registered at MycoBank Database (Crous et al. 2004; https://www.mycobank.org/, accessed 26 May 2023).

DNA extraction, PCR amplification, and Sequencing

Total genomic DNA was extracted from 7-d-old axenic cultures grown on PDA, according to the modified method of Boonyuen et al. (2021). The Internal transcribed spacers 1 and 2, including the intervening 5.8S nrDNA gene (ITS), were sequenced for every fungal isolate, and SSU, LSU, TEF-1α, TUB, ACT, RPB1 and RPB2 regions (White et al. 1990, Vilgalys & Hester 1990, Glass & Donaldson 1995, O’Donnell & Cigelnik 1997, Carbone & Kohn 1999, Liu et al. 1999, Voigt & Wöstemeyer 2000, Matheny et al. 2002, Castlebury et al. 2004, Rehner & Buckley 2005) were sequenced for each fungal strain that represented a new species candidate.

PCR was performed in a 50 µL reaction mixture containing 35.8 µL of nanopurified water, 5 µL of 10X Taq buffer with (NH₄)₂SO₄, 5 µL of 25 mM MgCl₂, 1 µL of 10 mM dNTP, 1 µL of each primer, 0.2 µL of recombinant Taq DNA polymerase (Thermo Scientific™) and 1 µL of fungal DNA. The primers ITS5/ITS4, ITS1F/4, NS1/NS4 (White et al. 1990), LROR/LR5 (Vilgalys & Hester 1990), Bt2a/Bt2b (Glass & Donaldson 1995), T1/T22 (O’Donnell & Cigelnik 1997), 983F/2218R (Rehner & Buckley 2005), tef1-728/tef1-1576 (Carbone & Kohn 1999), act1/act4 (Voigt & Wöstemeyer 2000), crpb1a/rpb1c (Castlebury et al. 2004) and RPB2-5F2/fRPB2-7cR (Liu et al. 1999) were employed. PCR profiles were obtained using the 100TM Thermal Cycler (BIO–RAD Laboratories, Inc., California) as described by Zhang et al. (2017, 2021). PCR products were checked by electrophoresis on 1 % agarose gels together with a DNA ladder containing DNA molecules of known sizes. To confirm the presence of amplicons at the expected molecular weight. PCR products were purified using a DNA purification kit (MACHEREY-NAGEL, Germany). The PCR products were used directly for DNA sequencing. DNA sequencing was performed at Macrogen Inc. in South Korea using the primers listed above. Forward and reverse reads were paired and consensus sequences, and they were checked for ambiguous bases and trimmed at both ends using BioEdit v. 7.2.3 (Hall 1999). The new sequences identified in this study were deposited in GenBank (National Center for Biotechnology Information; NCBI; accessed 26 May 2023). A BLASTn search of the GenBank database for the newly generated sequences was performed to exclude contamination and to search for related taxa (www.ncbi.nlm.nih.gov/blast; accessed 26 May 2023).

Sequence homologies were analysed using the BLAST search engine at NCBI and compared with the sequences reported in GenBank. If the ITS sequence of the highest BLAST hit for a given isolate had a closest similarity to potential genera and species of less than 97 %, the isolate was recognized as a potential novel species and was further identified using morphological data and multilocus phylogenetic analysis according to Zhang et al. (2017). The morphological data for potential novel species were compared with morphological data for known species identified to at least the genus or species level. Alignments were checked and manually optimised along with other sequences obtained from the GenBank nucleotide database.

Phylogenetic analysis

Multilocus phylogenetic analysis was conducted using the method of Boonyuen et al. (2011). Multiple alignments of the sequences generated from the novel species with other sequences obtained from GenBank were performed, and the results were refined manually in BioEdit v. 7.2.3 (Hall 1999), and automatically aligned using MUSCLE v. 3.8.1 (Edgar 2004). Manual gap adjustments were made to improve the alignment. Nine datasets were compiled in BioEdit v. 7.2.3, and the formats of multiple sequence alignments were changed using AliView (Larsson 2014). Bayesian posterior probabilities of the branches were obtained using MrBayes v. 3.2.6 (Ronquist et al. 2012) with the best-fit model (GTR+I+G) selected by AIC in MrModeltest v. 2.2 (Nylander 2004), which was tested with hierarchical likelihood ratios (hLRTs) and evaluated for the number of distinct data patterns under this model. Three million generations were run in four Markov chains and sampled every 100 generations with a burn-in value set at 3 000 sampled trees. Finally, maximum likelihood (ML) and bootstrap analyses were conducted using the CIPRES Science Gateway platform (Miller et al. 2010) with RAxML–HPC2 on XSEDE v. 8.2.8 (Stamatakis 2014) under the GTR + GAMMA model with the BFGS method to optimize GTR rate parameters. The resulting consensus tree was displayed using Interactive Tree Of Life (iTOL) (Letunic & Bork 2021;https://itol.embl.de/, accessed 26 May 2023) and adjusted in Adobe Photoshop 2020. All sequences generated in this study were deposited in GenBank and their respective accession numbers are given in Table 1. The resulting alignments were submitted to TreeBASE under submission IDs (30019, 29986, 29985, 29988, 29990, 29983, 29989, 29987 and 29984; https://www.treebase.org/treebase-web/home.html, accessed 26 May 2023).

Table 1.

Strain numbers and sequence accession numbers of eight novel species.

			GenBank accession numbers 2
Species name	Family	Strain number	ITS	LSU	SSU	TEF-1α	TUB	RPB1	RPB2	ACT
Actinomortierella caverna	Mortierellaceae	TBRC 162741	OP856536	OP856526	OP850837	NA	NA	NA	NA	NA
Hypoxylon phuphaphetense	Hypoxylaceae	TBRC 162771	OP856538	OP856528	NA	NA	OQ144973	NA	OQ108849	NA
Leptobacillium latisporum	Cordycipitaceae	TBRC 162881	OP856540	OP856529	OP850838	NA	NA	NA	NA	NA
Malbranchea phuphaphetensis	Malbrancheaceae	TBRC 162521	OP856532	OP856522	NA	OQ116929	OQ144969	NA	NA	NA
Scedosporium satunense	Microascaceae	TBRC 162851	OP856539	NA	NA	NA	OQ144974	NA	NA	NA
Sesquicillium cavernum	Bionectriceae	TBRC 162681	OP856535	OP856525	NA	OQ116931	OQ144971	NA	NA	NA
Thelonectria satunensis	Nectriaceae	TBRC 162751	OP856537	OP856527	NA	OQ116932	OQ144972	OQ076387	NA	OQ116938
Umbelopsis satunensis	Umbelopsidaceae	TBRC 162541	OP856533	OP856524	OP850836	NA	NA	NA	NA	OQ116937

¹Ex-holotype strains.

²NA = not available.

RESULTS

Phylogenetic analyses

The ITS phylogenetic analysis consists of 92 fungal taxa. Taphrina americana (CBS 331.55) and T. antarctica (CCFEE 5198) were used to root the tree (Fig. 4). Phylogenetic analyses showed that the eight novel species found in the karst cave (shown in red bold font in the figure) were distributed among eight families belonging to six different orders, i.e., Onygenales, Hypocreales, Microascales, Xylariales, Mortierellales and Umbelopsidales. The Bayesian inference (BI) tree is not shown because its topology is similar to that of the ML tree shown in the figure. Credible ML bootstrap values (≥ 50 %) and Bayesian posterior probabilities (≥ 0.95) are shown in the phylogenetic tree.

Fig. 4.

RAxML Phylogenetic tree of selected fungal groups showing the eight new species based on ITS region (90 ingroup taxa). Maximum likelihood bootstrap values (BSML, left) ≥ 50 % are shown at the nodes. Bayesian posterior probabilities (BYPP, right) ≥ 0.95 are given at the nodes. Highly supported nodes with 100 % bootstrap supports and with 1.00 posterior probabilities are shown as thick lines. Abbreviations: T = ex-holotype; ET = ex-epitype. Novel species are indicated by red bold font.

Taxonomy

Based on the morphological and molecular results provided above, several new species are proposed, namely Actinomortierella caverna, Hypoxylon phuphaphetense, Leptobacillium latisporum, Malbranchea phuphaphetensis, Scedosporium satunense, Sesquicillium cavernum, Thelonectria satunensis, and Umbelopsis satunensis.

Actinomortierella caverna C. Srihom, Preedanon, S. Saengkaewsuk & Somrith., sp. nov. MycoBank MB 846891. Fig. 5.

Fig. 5.

Actinomortierella caverna (TBRC 16274). A–C. Obverse and reverse views of cultures on PDA, MEA 2 % and OA after 14 d. D, F. Sporangiophore (arrow) with sporangiospores. E. uppermost branching of sporangiophore (arrow). G. Vesicle (arrow). H. sporangium wall (arrow) with sporangiospores. I. Sporangiospores. Scale bars: D–H = 20 µm; I =10 µm.

Etymology: “caverna” in Latin means “cave” and refers to the habitat in which this fungus was initially discovered.

Typus: Thailand, Satun Province, Thung Wa District, Satun UNESCO Global Geopark, Le Stegodon Cave, 7°7’35”N 99°59’49”E, 107 m elevation, isolated from soil, Dec. 2019, coll. N. Boonyuen, P. Srikitikulchai & S. Preedanon, isol., S. Preedanon, cultura dessicata (holotype BBH 49442; ex-type culture BCC 91669 = TBRC 16274 = NBTF 002290 = isolate CV00251). The GenBank accession numbers of ITS, LSU and SSU are OP856536, OP856526 and OP850837, respectively.

Classification: Mortierellaceae, Mortierellales, Incertae sedis, Mortierellomycetes, Mortierellomycotina, Mucoromycota

Description: Vegetative hyphae aseptate, 1.1–4.6 µm (n = 15) wide, smooth and hyaline. Sporangiophores arising from the mycelia, hyaline, 79.9–353 × 6.4–18.2 µm wide; branched sporangiophores forming sporangia, 7.6–60 × 2.2–4.8 µm wide. Sporangia globose to subglobose, 13–37.6 µm wide. Vesicle 56–60.1 µm diam. Sporangiospores hyaline, globose to subglobose, one-celled, 5.9–12.6 × 5.8–11.8 µm (n = 30),.

Culture characteristics: Colonies after 14 d on: PDA attaining 85 mm diam, umbonate, undulate, floccose, fluffy, margins curled, mycelia white (1A1); reverse yellowish white (1A2) to white (1A1) at margin. On MEA attaining 85 mm diam, filamentous, flat, margin rhizoids, striate, aerial mycelia extremely sparse and white (1A1); reverse white (1A1). On OA attaining 85 mm diam, convex, floccose, fluffy, tufts, dense and white (1A1), margin entire; reverse white (1A1). Sporulation within 9 wk on PDA.

Notes: Actinomortierella is characterised by sporangiophores with an inflated upper part bearing branches to form the sporangium (Chalabuda 1868, Degawa 1997, Degawa & Gams 2004, Vandepol et al. 2020). This genus is typically found in soils, manure, compost, decaying wood, and the rhizosphere (Seviour et al. 1987, Lorch et al. 2013, Macias et al. 2019). According to indexfungorum.org (accessed 26 May 2023), three species, A. ambigua, A. capitata, and A. wolfii, are currently accepted in the genus; these were initially described as Mortierella by Marchal (1891), Mehrotra et al. (1963), and Mehrotra & Baijal (1963), respectively. Based on phylogenetic analysis the new species belongs to Actinomortierella, and is related to A. capitata, (BSML = 99 %) (Fig. 6). However, the Bayesian posterior probability is less than 0.95. Additionally, comparison of the ITS barcode regions of A. capitata and A. caverna showed a sequence similarity of 85.81–89 %. Morphologically, A. caverna branches at the uppermost portion of the sporangiophore, but the inflation part from which the branches arise is rarely observed. Additionally, the new species has only a few branches, unlike the other two species. Actinomortierella capitata has sporangia that are much larger than those of the new species (Supplementary Table S1). Actinomortierella caverna differs from A. ambigua and A. wolfii in the shape of its spores. Spores of A. caverna are globose to subglobose, while spores of A. ambigua are oblong, and those of A. wolfii are ellipsoid-reniform. Distinctions between the novel taxa and their close relatives are presented in Supplementary Table S1. The new species was discovered in the cave habitat for the first time. Morphological characteristics and phylogenetic analyses of combined ITS, LSU and SSU sequences support the new species and the placement of A. caverna in the Mortierellaceae.

Fig. 6.

RAxML phylogenetic tree of Actinomortierella caverna (TBRC 16274) and related taxa resulting from the combination of ITS, LSU and SSU sequences (57 ingroup taxa). Maximum likelihood bootstrap values (BSML, left) ≥ 50 % are shown at the nodes. Bayesian posterior probabilities (BYPP, right) ≥ 0.95 are given at the nodes. Highly supported nodes with 100 % bootstrap supports and with 1.00 posterior probabilities are shown as thick lines. Abbreviations: T = ex-holotype; ST = ex-syntype; NT = ex-neotype; IT = ex-isotype. Novel species is shown in red bold font.

Hypoxylon phuphaphetense C. Srihom, Preedanon, S. Saengkaewsuk & Somrith., sp. nov. MycoBank MB 846894. Fig. 7.

Fig. 7.

Hypoxylon phuphaphetense (TBRC 16277). A–C. Obverse and reverse views of cultures on PDA, MEA 2 %, and OA after 14 d after. D. Conidiophores with conidia. E–G. Conidiogenous cells with conidia. H. Conidia. Scale bars: D–H = 10 µm.

Etymology: “phuphaphetense”, pertaining to Phu Pha Phet cave in which the fungus was first discovered.

Typus: Thailand, Satun Province, Manang District, Satun UNESCO Global Geopark, Phu Pha Phet Cave, 7°7’25”N 99°47’54”E, 21 m elevation, isolated from soil, Dec. 2019, coll. N. Boonyuen, P. Srikitikulchai & S. Preedanon, isol., S. Preedanon, cultura dessicata (holotype BBH 49444; ex-type culture BCC 91956 = TBRC 16277 = NBTF 002284, isolate CV00283). The GenBank accession numbers of ITS, LSU, RPB2 and TUB are OP856538, OP856528, OQ108849, and OQ144973, respectively.

Classification: Hypoxylaceae, Xylariales, Xylariomycetidae, Sordariomycetes, Pezizomycotina, Ascomycota.

Description: Conidiophores micro- to semi-macronematous, hyaline, simple or branched, smooth to slightly roughened, 1.7–17 × 0.8–2.2 µm. Conidiogenous cells holoblastic, mono-or polyblastic, aggregated, hyaline to brown, straight to curved with scars. Conidia ellipsoid, pale brown to dark brown, smooth-to rough-walled, 5–10.2 × 4–5 µm (n = 30) with protruding hilum, more abundant on PDA than on 2 % MEA or on OA. Sexual morph not observed.

Culture characteristics: Colonies after 14 d on: PDA attaining 60 mm diam, circular, flat, felt, annular, margin radially striate with lobate edge and greyish brown (5F3) to orange grey (5B2) at margin; reverse dark brown (6F8) to reddish yellow (4A6) at margin. On 2 % MEA attaining 40 mm diam, circular, flat, entire, more intense near the medium surface, sparse and greyish brown (5F3); reverse yellowish brown (5D8). On OA attaining 60 mm diam, irregular, flat, undulate, and more intense near the medium surface, dull and brownish grey (5F2); reverse greyish yellow (4B3). Sporulation within 6 wk on PDA.

Notes: The genus Hypoxylon within the family Hypoxylaceae (Xylariales, Ascomycota) was proposed by Bulliard (1791). More than 200 species have been recorded (Becker et al. 2020, Pourmoghaddam et al. 2020, Cedeño-Sanchez et al. 2023). It is a common fungus in natural forests, and some species of the genus reside in dead plants or as endophytes, while others are beneficial and play important roles in the ecosystem (Dayarathne et al. 2020, Hyde et al. 2020, Ma et al. 2022, Song et al. 2022).

Phylogenetically, H. phuphaphetense forms a sister clade with H. hinnuleum in Hypoxylon clade H1 with BSML = 100 % and BYPP = 1.00, as shown in Fig. 8. However, these two clades appear highly different based on their morphological data. Hypoxylon hinnuleum was proposed as the sexual morph of Nodulisporium hinnuleum (Sir et al. 2019). The asexual morph of H. hinnuleum is Virgariella or Nodulisporium, with a long conidiophore with branches and a denticulate conidiogenous cell. Smith (1962) first described Nodulisporium hinnuleum from sorghum and oat in Kansas, USA, as a culture contaminant. Later, Sir et al. (2019) collected a number of Hypoxylon specimens in Texas, USA and considered N. hinnuleum as their asexual morph. In contrast, H. phuphaphetense is unique in that it only possesses an asexual morph. It produces a simple or clustered conidiogenous cell that is short and lacks denticulation (small tooth-like projections). Its asexual morphology differs significantly from the typical asexual morphs observed in the genus, which usually exhibit characteristics resembling Nodulisporium. Furthermore, the conidia of H. hinnuleum are hyaline and thin-walled, while those of the new species are pale to dark brown and relatively thick-walled. Additionally, the conidia of the new species are markedly larger (4.9–10.2 × 4.1–5.6 µm in H. phuphaphetense vs. 3.6–5.1 × 2.1–3.1 µm in H. hinnuleum). The ITS barcode region of H. phuphaphetense was compared with those of H. hinnuleum (MUCL 3621, DSM 107932, DSM 107926) sequences, and the results showed 494/564 = 89.36 % similarity with 10 substitutions, 491/563 = 89.34 % similarity with 10 substitutions and 493/565 = 89.03 % similarity with 10 substitutions, respectively. Based on the observed morphology and phylogeny, we introduce our karst cave collection as a new taxon.

Fig. 8.

The RAxML phylogenetic tree of Hypoxylon phuphaphetense (TBRC 16277) and related taxa based on sequences at four loci (ITS, LSU, RPB2 and TUB) consists of 53 ingroup taxa. Maximum likelihood bootstrap values (BSML, left) ≥ 50 % are shown at the nodes. Bayesian posterior probabilities (BYPP, right) ≥ 0.95 are given at the nodes. Highly supported nodes with 100 % bootstrap supports and with 1.00 posterior probabilities are shown as thick lines. Abbreviations: T = ex-holotype; ET = ex-epitype. Novel species is shown in red bold font. H1 represents a sister clade of Hypoxylon spp., in which our new species is closed.

Leptobacillium latisporum C. Srihom, Preedanon, S. Saengkaewsuk & Somrith., sp. nov. MycoBank MB 846897. Fig. 9.

Fig. 9.

Leptobacillium latisporum (TBRC 16288). A–C. Obverse and reverse views of cultures on PDA, MEA 2 %, and OA after 14 d. D. Conidiophores with conidia. E, F. Chains of conidia. Scale bars: D = 15 µm; E, F = 5 µm.

Etymology: “latis” from Latin, lati- means broad, and “sporum” means spores or conidia, referring to the broad conidia of this species compared with those of other species.

Typus: Thailand, Satun Province, Manang District, Satun UNESCO Global Geopark, Phu Pha Phet Cave, 7°7’25”N 99°47’54”E, 21 m elevation, isolated from soil, Dec. 2019, coll. N. Boonyuen, P. Srikitikulchai & S. Preedanon, isol., S. Preedanon, cultura dessicata (holotype BBH 49446; ex-type culture BCC 91807 = TBRC 16288 = NBTF 002288 = isolate CV00353). The GenBank accession numbers of ITS, LSU, and SSU are OP856540, OP856529 and OP850838, respectively.

Classification: Cordycipitaceae, Hypocreales, Hypocreomycetidae, Sordariomycetes, Pezizomycotina, Ascomycota.

Description: Vegetative hyphae hyaline, 1.2–3.6 µm wide (n = 15), Conidiophores macronematous, mostly solitary, cylindrical. Conidiogenous cells phialide, often branched with two or three phialides which being furcate (= schizophialides), hyaline, 13.2–40.8 × 3–4.8 µm. Conidia mostly formed in a long dry chain, slightly fusoid to narrowly cylindrical, 4–6.3 × 1.9–3.8 µm. Chlamydospores and sexual morph not observed.

Culture characteristics: Colonies after 14 d on: PDA attaining 45 mm diam, circular, raised, entire, velvety, fluffy, dense, delicate and white (5A1); reverse smooth with indistinct radial furrows, greyish orange (5B4) to orange white (5A2) at margin. On MEA attaining 70 mm diam, circular, raised, entire, velvety, fluffy, dense, delicate and white (5A1); reverse smooth with indistinct radial furrows, brownish orange (5C4) to orange white (5A2) at margin. On OA attaining 45 mm diam, circular, raised, entire, velvety, fluffy, dense, delicate and white (5A1); reverse orange white (5A2). Sporulation within 6 wk on PDA.

Notes: The genus Leptobacillium was proposed by Zare & Gams (2016) with L. leptobactrum as the type species. This genus is a member of the Cordycipitaceae. The genus is characterised by fusoid to cylindrical conidia born in chains and accumulated at the tip of a phialide that is usually solitary on hyphae. Molecular phylogeny supports the segregation of Leptobacillium from Simplicillium, a closely related genus (Chen et al. 2021). Based on indexfungorum.org (accessed 26 May 2023), Leptobacillium currently includes seven species. Recently, L. cavernicola from palaeolithic-decorated caves in France was described (Leplat et al. 2022), and L. symbioticum and L. leptobactrum were found in air samples from show caves in Spain (Dominguez-Moñino et al. 2021), India (Phookamsak et al. 2019) and Japan (Okane et al. 2020). In this study, the new species was identified as Leptobacillium based on its morphological and phylogenetic data. However, the new species differs markedly from other species in having the widest conidia. Furthermore, the conidiophores of the new species are often branched with two or three phialides, while the conidiophores of other species are mainly solitary phialides. Phylogenetic analysis also supports the distinction because the new species forms a sister clade of the cluster of the described species, with strong support (MSML= 92 % and BYPP = 1.00) (Fig. 10). Morphological comparisons between the new species and closely related Leptobacillium are provided in Supplementary Table S2. Our fungus represents a new karst cave taxon that is morphologically and phylogenetically distinguished from other species, and we propose it L. latisporum.

Fig. 10.

RAxML phylogenetic tree of Leptobacillium latisporum (TBRC 16288) and related taxa resulting from the combined datasets of ITS, LSU and SSU sequences (48 ingroup taxa). Maximum likelihood bootstrap values (BSML, left) ≥ 50 % are shown at the nodes. Bayesian posterior probabilities (BYPP, right) ≥ 0.95 are given at the nodes. Highly supported nodes with 100 % bootstrap supports and with 1.00 posterior probabilities are shown as thick lines. Abbreviation: T = ex-holotype. Novel species is shown in red bold font.

Malbranchea phuphaphetensis C. Srihom, Preedanon, S. Saengkaewsuk & Somrith., sp. nov. MycoBank MB 846887. Fig. 11.

Fig. 11.

Malbranchea phuphaphetensis (TBRC 16252). A–C. Obverse and reverse views of cultures on PDA, MEA 2 %, and OA after 14 d. D, E. Intercalary arthroconidia (arrow) along the fertile hyphae. F–I. Arthroconidia. Scale bars D, E = 5 µm; F–I = 10 µm.

Etymology: “phuphaphetensis” refers to the name of the cave in which the fungus was first discovered.

Typus: Thailand, Satun Province, Manang District, Satun UNESCO Global Geopark, Phu Pha Phet Cave, 7°7’25”N 99°47’54”E, 21 m elevation, isolated from soil, Dec. 2019, coll. N. Boonyuen, P. Srikitikulchai & S. Preedanon, isol., S. Preedanon, cultura dessicata (holotype BBH 49439; ex-type culture BCC 91900 = TBRC 16252 = NBTF 002285 = isolate CV 00115). The GenBank accession numbers of ITS, LSU, BenA and TEF-1α are OP856532, OP856522, OQ144969 and OQ116929, respectively.

Classification: Onygenaceae, Onygenales, Eurotiomycetidae, Eurotiomycetes, Pezizomycotina, Ascomycota.

Description: Vegetative hyphae hyaline, septate, 1.5–3.6 µm wide (n = 15). Arthroconidia formed from narrow hyphae, one-celled, hyaline smooth and thin-walled, cylindrical or short-cylindrical, straight, 4.2–9.4 × 2.4–4.5 µm (n = 30). Chlamydospores and sexual morph not observed.

Culture characteristics: Colonies after 14 d on: PDA attaining 35 mm diam, circular, flat, entire, more intense near the medium surface, velutinous and deep orange (6A8) to light yellow (4A5) at margin; reverse deep orange (6A8) to light yellow (4A5) at margin. On MEA attaining 25–30 mm diam, circular, flat, entire, velutinous, and dark orange (5A8) to pale yellow (4A3) at margin; reverse dark orange (5A8) to pale yellow (4A3) at margin. On OA attaining 30 mm diam, circular, flat, entire, woolly and brownish orange (5C5) to light yellow (4A4) at margin; reverse greyish orange (5B3). Sporulation within 9 wk on PDA.

Notes: Saccardo (1882) proposed Malbranchea with M. pulchella as the type species. The genus is characterised by alternate arthroconidia originating from vegetative hyphae in its asexual morph. In its sexual morph, it exhibits distinct features including orange to brown coloration, appendages, and/or spines. It forms gymnothecial ascomata, which are structures that enclose the reproductive organs. These ascomata have a prototunicate structure, meaning they consist of a single layer. Within the ascomata, the genus produces eight-spored inflated asci, which are sac-like structures. These asci contain globose to oblate ascospores that have a reticulate pattern on their surface. Based on indexfungorum.org (accessed 26 May 2023), 43 species are presently referred to the genus Malbranchea, and 42 species have sequences of at least one locus in GenBank. Phylogenetic analysis based on ITS, LSU, BenA and TEF-1α sequences showed that our isolate clustered within the genus Malbranchea (Fig. 12). The new taxon, M. phuphaphetensis is phylogenetically related to M. ostraviensis, which was originally described as Auxarthron ostraviense by Hubka et al. (2013), and M. gymnoascoides (Rodríguez-Andrade et al. 2021) (Fig. 12). The ITS barcode region of M. phuphaphetensis was compared with those of M. ostraviensis and M. gymnoascoides, and the results showed 416/459 = 92.37 % similarity with 8 substitutions and 414/459 = 92.16 % similarity with 9 substitutions, respectively. These species have similar orange shade of colony colour (Rodríguez-Andrade et al. 2021, Kandemir et al. 2022). However, M. phuphaphetensis has a much longer arthroconidium (4.2–19.4 × 2.4–4.5 µm) than M. gymnoascoides (1.5–2 × 6–10 µm) and M. ostraviensis (1.5–2.5 × 3.8–12.6 µm). Therefore, we propose M. phuphaphetensis as new species.

Fig. 12.

RAxML phylogenetic tree of Malbranchea phuphaphetensis (TBRC 16252) and related taxa based on ITS, LSU, BenA and TEF-1α sequences (56 ingroup taxa). Maximum likelihood bootstrap values (BSML, left) ≥ 50 % are shown at the nodes. Bayesian posterior probabilities (BYPP, right) ≥ 0.95 are given at the nodes. Highly supported nodes with 100 % bootstrap supports and with 1.00 posterior probabilities are shown as thick lines. Abbreviations: T = ex-holotype; NT = ex-neotype. Novel species is shown in red bold font.

Scedosporium satunense C. Srihom, Preedanon, S. Saengkaewsuk & Somrith., sp. nov. MycoBank MB 846896. Fig. 13.

Fig. 13.

Scedosporium satunense (TBRC 16285). A, B. Obverse and reverse views of cultures on PDA and CMA after 14 d. C, E. Conidiophores. D, F. Phialides with conidia. G, H. Conidia. Scale bars: D = 5 µm; C, E–H = 10 µm.

Etymology: “satunense” was named after the province from which this fungus was initially collected.

Typus: Thailand, Satun Province, Thung Wa District, Satun UNESCO Global Geopark, Le Stegodon Cave, 7°7’35”N 99°59’49”E, 107 m elevation, isolated from soil, Dec. 2019, coll. N. Boonyuen, P. Srikitikulchai & S. Preedanon, isol., S. Preedanon, cultura dessicata, (holotype BBH 49445; ex-type culture BCC 91803 = TBRC 16285 = NBTF 002291 = isolate CV00322). The GenBank accession numbers of ITS and BenA are OP856539 and OQ144974, respectively.

Classification: Microascaceae, Microascales, Hypocreomycetidae, Sordariomycetes, Pezizomycotina, Ascomycota.

Description: Vegetative hyphae hyaline to pale brown, septate, branched, smooth or slightly rough, hyphae 1.7–3.3 µm wide. Conidiophores macronematous, hyaline, solitary, frequently reduced to a single conidiogenous cell arising laterally from hypha, or forming short-stalked, penicilloid, 10.2–33.2 × 1.4–3.1 µm, bearing two or three conidiogenous cells at the top. Conidiogenous cells annellidic, hyaline, smooth and thin-walled, cylindrical, 3.8–14.7 × 1.7–3.2 µm. Conidia enteroblastic, one-celled, solitary, hyaline, ellipsoidal to cylindrical, 5.7–10.5 × 4.1–5.6 µm, brown, smooth and thick-walled. Sexual morph not observed.

Culture characteristics: Colonies after 14 d on: PDA attaining 60 mm diam, circular, umbonate, filiform, dense, felt, smooth with indistinct radial furrows and grey (1D1) to white (1A1) at the margin; reverse medium grey (1E1) to white (1A1) at margin. On CMA attaining 50 mm diam, circular, flat, filiform, aerial mycelia extremely sparse and greyish brown (5D3); reverse dark blond (5D4) to white (5A1) at margin. Sporulation within 6 wk on PDA.

Notes: The genus Scedosporium, designed by Sacc. ex Castell. & Chalm. (1919), includes saprobic fungi, with a few species reported to cause disease in humans (de Hoog et al. 2000, Rainer & de Hoog 2006, Lackner et al. 2014, Abrantes et al. 2021). Scedosporium is characterised by cylindrical to flask-shaped and annellidic conidiogenous cells borne in solitary on the hyphae or in penicillate groups on the conidiophore. The genus is also reported to include the morphology of the pseudallescheria-like sexual morph, which is characterised by fusiform one-celled ascospores borne in an oval or spherical ascus inside the cleistothecia (Tapia 2012, Lackner et al. 2014). Molecular phylogeny usually assists in species separation (Gilgado et al. 2008, Lackner et al. 2012, Subedi & Chen 2015). In our findings, S. satunense clusters together with S. americanum (96 % ML bootstrap proportion and 1.00 Bayesian posterior probability) (Fig. 14). In particular, the ITS rDNA sequence of S. satunense was compared with ITS rDNA sequences in S. americanum (CBS 218.35 and DMic 165285); the results showed 523/555 = 94.23 % similarity with no substitution and 530/556 = 95.32 % similarity with no substitution, respectively. Analysis of the BenA gene showed that there were base substitutions at multiple positions, resulting in 94.25 % similarity with nine substitutions. The new species differs from S. americanum in several aspects. For example, colonies of the new species are shades of white to grey and grow faster (reaching diameters of approximately 60 mm in 14 d), while those of S. americanum are brown to olive-brown and reach diameters of approximately 30 mm in 14 d (Abrantes et al. 2021). The conidia of S. americanum are ellipsoid to clavate and smaller (5.4–6.8 × 4.2–4.8 µm, Abrantes et al. 2021) than those of the new species, which are ellipsoid to oblong and slightly larger (5.8–10.5 × 4.1–5.6 µm). Additionally, synnemata are not present in S. satunense, while they are clearly observed in S. americanum (Abrantes et al. 2021). Both morphological and DNA sequence data support the distinction of S. satunense as a novel karst cave taxon in this study.

Fig. 14.

he RAxML phylogenetic tree of Scedosporium satunense (TBRC 16285) and related taxa based on sequences at two loci (ITS and BenA) consists of 48 ingroup taxa. Maximum likelihood bootstrap values (BSML, left) ≥ 50 % are shown at the nodes. Bayesian posterior probabilities (BYPP, right) ≥ 0.95 are given at the nodes. Highly supported nodes with 100 % bootstrap supports and with 1.00 posterior probabilities are shown as thick lines. Abbreviations: T = ex-holotype; NT = ex-neotype. Novel species is shown in red bold font.

Sesquicillium cavernum C. Srihom, Preedanon, S. Saengkaewsuk & Somrith., sp. nov. MycoBank MB 846889. Fig. 15.

Fig. 15.

Sesquicillium cavernum (TBRC 16268). A, B. Obverse and reverse views of cultures on PDA and OA after 14 d. C, F. Penicillate conidiophores with phialides and conidia. D, E, G. Chlamydospores. H, I. Conidia. Scale bars: C = 20 µm; D, E, G–I = 10 µm; F = 5 µm.

Etymology: “cavernum” in Latin means “cave” and refers to the habitat in which this species was first discovered.

Typus: Thailand, Satun Province, Thung Wa District, Satun UNESCO Global Geopark, Le Stegodon Cave, 7°7’35”N 99°59’49”E, 107 m elevation, isolated from shells of dead snails, Dec. 2019, coll. N. Boonyuen, P. Srikitikulchai & S. Preedanon, isol., S. Preedanon, cultura dessicata (holotype BBH 49441; ex-type culture BCC 91623 = TBRC 16268 = NBTF 002289 = isolate CV00218). The GenBank accession numbers of ITS, LSU, TUB and TEF-1α are OP856535, OP856525, OQ144971 and OQ116931, respectively.

Classification: Bionectriaceae, Hypocreales, Hypocreomycetidae, Sordariomycetes, Pezizomycotina, Ascomycota.

Description: Vegetative hyphae hyaline to pale brown, septate, smooth, 1.2–3.2 µm wide. Conidiophores hyaline to pale brown, septate, short and solitary or penicillate branched; conidiophores from aerial hyphae were septate with dense phialides, 20.1 × 33–36 µm. Phialides subulate, tapering towards the apex, 10.2–12.5 µm long, 1.5–4 µm wide at the base, and 0.2–0.3 µm wide at the tip, intercalary phialides are rarely observed. Conidia enteroblastic, one-celled, solitary, hyaline, subglobose to broadly ellipsoidal, 3.9–6 × 2.3–4.6 µm, smooth and thick-walled. Chlamydospores solitary, hyaline, intercalary, broadly ellipsoidal, smooth, 6.0–7.5 × 5.0–6.3 µm wide. Sexual morph not observed.

Culture characteristics: After 14 d Colonies on PDA attaining 35 mm diam, circular, raised, entire, velvety, dense, delicate and orange, white (5A2) to white (5A1) at margin; reverse light orange (5A4) to orange, white (5A2) at the margin. On OA attaining 45–55 mm diam, circular, umbonate, entire, mycelia extremely tufted at the centre, margin annular and white (3A1); reverse yellowish white (3A2). Sporulation within 6 wk on PDA.

Notes: Sesquicillium was erected by Gams (1968). Recently, Zhao et al. (2023) revealed that Clonostachys and Sesquicillium are closely related and constitute sister clades based on multiple gene phylogenetic analyses. Furthermore, some species of Clonostachys were allocated to Sesquicillium when the genus was revised. Morphologically, there is little differentiation of the asexual morphs (i.e., penicillium-, verticillium-, gliocladium-, or acremonium-like conidiophores) among the species; thus, molecular phylogeny is usually employed for species identification (Corda 1839, Moreira et al. 2016, Rossman et al. 2013, Torcato et al. 2020, Zeng & Zhuang 2022, Zhao et al. 2023). In this study, S. cavernum was found to be distantly related to S. candelabrum (originally Verticillium candelabrum according to Bonorden 1851) and S. rossmaniae with BSML = 71 % and BYPP = 1.00 (Fig. 16). Sesquicillium cavernum differs from those two species in having relatively wider conidia (3.9–6 × 2.3–4.6 µm in S. cavernum vs. 3–5.5 × 1.8–3.4 µm in S. candelabrum and 4.2–6.6 × 2–2.8 µm in S. rossmaniae). The morpho-phylogenetic evidence presented here allow us to describe S. cavernum as a new species that occurs on shells of dead snails inside a cave in Thailand.

Fig. 16.

The RAxML phylogenetic tree of Sesquicillium cavernum (TBRC 16268) and related taxa based on sequences at four loci (ITS, LSU, TUB and TEF-1α) consists of 43 ingroup taxa. Maximum likelihood bootstrap (BSML, left) values ≥ 50 % are shown. Bayesian posterior probabilities (BYPP, right) ≥ 0.95 are given at the nodes. Highly supported nodes with 100 % bootstrap supports and with 1.00 posterior probabilities are shown as thick lines. Abbreviations: T = ex-holotype; IT = ex-isotype; NT = ex-neotype. Novel species is shown in red bold font.

Thelonectria satunensis Chuaseehar., Nuankaew, Preedanon & Somrith., sp. nov. MycoBank MB 846893. Fig. 17.

Fig. 17.

Thelonectria satunensis (TBRC 16275). A, B. Obverse and reverse views of cultures on PDA and 1/4–strength PDA after 21 d. C. Perithecia produced on 1/4–strength PDA. D. Longitudinal section of perithecium. E, F. Asci and ascospores. G. Periphyses and asci with ascospores. H. Conidiophores, phialides and macroconidia. I, J. Macroconidia. Scale bars: C, D = 100 µm; E, H–J = 20 µm; F, G = 10 µm.

Etymology: “satunensis” meaning “derived from Satun”, the province in which this fungus was first discovered.

Typus: Thailand, Satun Province, Manang District, Satun UNESCO Global Geopark, Phu Pha Phet Cave, 7°7’25”N 99°47’54”E, 21 m elevation, airborne, Dec. 2019, coll. N. Boonyuen, P. Srikitikulchai & S. Preedanon, isol., S. Preedanon, cultura dessicata (holotype BBH 49443; ex-type culture BCC 91755 = TBRC 16275 = NBTF 002337 = isolate CV00255). The GenBank accession numbers of ACT, ITS, LSU, RPB1, TEF-1α and TUB are OQ116938, OP856537, OP856527, OQ076387, OQ116932, and OQ144972, respectively.

Classification: Nectriaceae, Hypocreales, Hypocreomycetidae, Sordariomycetes, Pezizomycotina, Ascomycota.

Description: Ascomata on 1/4-strength PDA after 30 d perithecial, solitary to gregarious, up to five in a group, superficial, sometimes immersed in agar medium or with the base immersed in agar medium on a minute stroma, pyriform to subglobose, papillate, yellowish-brown to brown, no colour change in 3 % KOH or 100 % lactic acid, 350–500 × 350–370 μm (n = 10). Perithecial surface smooth or slightly roughened. Perithecial wall lacking a definite outline, appearing to be intertwined hyphae with angular cells and flattened fusoid cells, 29–39 µm thick. Asci unitunicate, cylindrical to clavate, 8-spored, 57–88 × 10–13 μm (n = 30), with a refractive ring at the apex. Ascospores uniseriate, ellipsoid, finely spinulose, 1-septate, symmetrically two-celled, initially hyaline, becoming pale brown at maturity, 10–17.5 × 7.5 µm (n = 30). Asexual morph: Mycelia smooth, branched, septate, hyaline hyphae, pale brown, and 1.5–4.5 μm in diam. Conidiophores arising laterally from hyphae, unbranched to diverticillate branched, septate, 19–33 × 2.5–4.5 μm (n = 10). Phialides cylindrical or ampulliform, borne solitary or apically on irregularly branching clusters of cells or directly from hyphae, 7–18 × 2.5–4.5 μm (n = 10), with periclinal thickening and collarette. Conidia hyaline, slimy droplets on aerial mycelium or arising from the agar surface. Macroconidia cylindrical or falcate, curved, rounded on both ends, smooth, 3–5-septate, hyaline, 27.5–45 × 7.5–10 μm (n = 30). Chlamydospores and microconidia not observed.

Culture characteristics: Colonies after 21 d on: PDA reaching 64–69 mm diam, irregular, raised, margins curled, mycelia white, floccose; sporulation absent, soluble pigment absent, exudates absent; reverse yellowish white (1A2) with margins white. On 1/4-strength PDA attaining 55–56 mm diam, irregular, flat, margins curled, mycelia white with centre yellowish brown (5D8), mostly glabrous surface with sparsely aerial hyphae; sporulation producing asexual morph moderately and ascomata sparsely after 30 d of incubation, conidia in mas white, soluble pigment absent, exudates absent; reverse white with centre yellowish brown (5D8).

Notes: Thelonectria, previously placed in the genus Neonectria, is a recently established genus of common and ubiquitous fungi that is found on living and decaying woody substrates, soil, other fungi, and insects (Salgado-Salazar et al. 2012, Zeng & Zhuang 2019, Crous et al. 2022b, Zeng & Zhuang 2022). In the phylogenetic analysis, T. satunensis grouped as a separate clade with T. asiatica, T. brayfordii, T. conchyliata, T. discophora, T. guangdongensis, T. japonica, T. lucida, T. mammoidea, T. ostrina, T. phoenicea, T. pinea, T. porphyria, T. purpurea, T. rubi, T. sinensis and T. yunnanica. In the phylogenetic analysis, placement of this new species in a major clade in the Nectriaceae (Hypocreales) was strongly supported with 100 % ML bootstrap and 1.00 BI posterior probabilities. In this study, T. satunensis is well separated from the other species, with a number of nucleotide base substitutions in ACT (97.40–97.77 %), ITS (93.80–94.60 %), LSU (97.89–98.26 %), RPB1 (89.37–90 %), TEF-1α (92.67–94.11 %), and TUB (91.98–92.75 %), as shown in Fig. 18. Among the known phylogenetically related species, T. satunensis is morphologically similar to T. ostrina and T. porphyria; these three species share papillate perithecia and 3–5-septate macroconidia (Salgado-Salazar et al. 2015). However, T. satunensis differs from those two species in forming white colonies and having shorter macroconidia (27.5–45 × 7.5–10 μm), while T. ostrina and T. porphyria form purple colonies and have longer conidia (40–108 × 5–9 μm in T. ostrina and 42.5–88.5 × 4.5–7.5 μm in T. porphyria; Salgado-Salazar et al. 2015). Thus, T. satunensis is newly introduced on the basis of its morphological characteristics and on DNA sequence analysis of combined datasets of its ACT, ITS, LSU, RPB1, TEF-1α and TUB sequences.

Fig. 18.

The RAxML phylogenetic tree of Thelonectria satunensis (TBRC 16275) and related taxa based on the sequences from six loci (ACT, ITS, LSU, RPB1, TEF-1α and TUB) consisting of 44 ingroup taxa. Maximum likelihood bootstrap values (BSML, left) ≥ 50 % are shown at the nodes. Bayesian posterior probabilities (BYPP, right) ≥ 0.95 are given at the nodes. Highly supported nodes with 100 % bootstrap supports and with 1.00 posterior probabilities are shown as thick lines. Abbreviations: T = ex-holotype. Novel species is shown in red bold font.

Umbelopsis satunensis C. Srihom, Preedanon, S. Saengkaewsuk & Somrith., sp. nov. MycoBank MB 846888. Fig. 19.

Fig. 19.

Umbelopsis satunensis (TBRC 16254). A–C. Obverse and reverse views of cultures on PDA, MEA 2 %, and CMA after 14 d. D. Chlamydospores. E, F. Immature sporangia with sporangiospores (arrow). G. Mature sporangium with angular sporangiospores. H. Sporangiospores in the angular shape. Scale bars: D–F = 20 µm; G, H = 10 µm.

Etymology: “satunensis” refers to “Satun province”, from which the ex-type strain was collected.

Typus: Thailand, Satun Province, Thung Wa District, Satun UNESCO Global Geopark, Le Stegodon Cave, 7°7’35”N 99°59’49”E, 107 m elevation, isolated from shells of dead snails, Dec. 2019, coll. N. Boonyuen, P. Srikitikulchai & S. Preedanon, isol., S. Preedanon, cultura dessicata, (holotype BBH 49440; ex-type culture BCC 91926 = TBRC 16254 = NBTF 002286 = isolate CV00129). The GenBank accession numbers of SSU, ITS, LSU and ACT are OP856533, OP856524, OP850836 and OQ116937, respectively.

Classification: Umbelopsidaceae, Umbelopsidales, Incertae sedis, Umbelopsidomycetes, Mucoromycotina, Mucoromycota.

Description: Vegetative hyphae, smooth, hyaline 1.2–3.6 µm (n = 15) wide, Sporangiophores developing from abundant aerial hyphae, hyaline, smooth, with compactly sympodial branching from slightly swollen stalks, 2.2–4 µm wide, up to 125 µm long. Sporangia at tips of sporangiophores globose, thin-walled and double-layered, 22.7–34 µm in diam. Sporangiospores smooth, angular, 3.6–4.0 × 3.3–3.9 µm in diam (n = 30). Chlamydospores hyaline, smooth, globose to subglobose, 6.3–6.8 × 6.5–6.9 µm in diam. Zygospores not observed.

Culture characteristics: Colonies after 14 d on: PDA attaining 75 mm diam, circular, flat, entire, more intense near the medium surface, sparse, butter-like and vivid yellow (3A8) to yellowish white (3A2) at margin; reverse vivid yellow (3A8) to yellowish white (3A2). On MEA attaining 75 mm diam, circular, flat, entire, more intense near the medium surface, membranous and pale yellow (3A3) to yellowish white (3A2); reverse yellowish white (3A2). On CMA attaining 85 mm diam, circular, flat, entire, more intense near the medium surface, membranous, butter-like and pale yellow (3A3); reverse pale yellow (3A3). Sporulation within 6 wk on PDA.

Notes: Umbelopsis (Umbelopsidaceae, Umbelopsidales, Mucoromycota) was initially described by Amos & Barnett (1966) with U. versiformis as type species. The MycoBank database currently recognises 25 species in this genus (https://www.mycobank.org, accessed 26 May 2023). Environmentally, Umbelopsis species are a common and important constituent of forest soils (Domsch et al. 1980, Parshikov et al. 1999, Summerbell 2005, Wang et al. 2015, Ikeda et al. 2016).

Based on phylogenetic analysis (Fig. 20), Umbelopsis satunensis is well placed in the genus Umbelopsis and forms a sister clade with U. dura, U. macrospora and U. oblongielliptica. These three species share morphological characteristics in that they lack vesicles from which the sporangiophores develop. However, U. satunensis differs markedly in that it produces spores that are angular in shape, while the spores of U. macrospora and U. oblongielliptica are oblong-ellipsoidal, and those of U. dura are ovoid. Umbelopsis satunensis is further distinguished from other species in this genus by the relatively larger sizes of its sporangium and sporangiospores. The spores of U. satunensis are up to 7 µm diam, while the spores of U. dura, U. macrospora and U. oblongielliptica range from 2.8 to 5 µm in length and 1.6 to 2.8 µm in width. In addition, based on DNA sequence U. satunensis differs from other related species like U. dura, U. macrospora and U. oblongielliptica sequences in that it displays a number of nucleotide base substitutions in SSU (99.43–99.52 %), ITS (94.83–95.16 %), LSU (98.62–99.04 %), and ACT (95.37–95.87 %).

Fig. 20.

RAxML phylogenetic tree of Umbelopsis satunensis (TBRC 16254) and related taxa based on sequences from four loci (SSU, ITS, LSU and ACT) consisting of 43 ingroup taxa. Maximum likelihood bootstrap values (BSML, left) ≥ 50 % are shown at the nodes. Bayesian posterior probabilities (BYPP, right) ≥ 0.95 are given at the nodes. Highly supported nodes with 100 % bootstrap supports and with 1.00 posterior probabilities are shown as thick lines. Abbreviations: T = ex-holotype; ET = ex-epitype; IT = ex-isotype. Novel species is shown in red bold font.

DISCUSSION

Our research on cave-dwelling fungi in the Satun UNESCO Global Geopark in southern Thailand revealed eight new species which are described in this study (Ascomycota and Mucoromycota). Approximately 1 600 species of fungi have been recorded from caves worldwide (Vanderwolf et al. 2013, Jiang et al. 2017, Zhang et al. 2017, 2020, Visagie et al. 2021); the most species-rich phylum is Ascomycota, followed by Basidiomycota and Mucoromycota. The common genera belonging to the Ascomycota are mostly cosmopolitan, i.e., Aspergillus, Penicillium, Fusarium and Trichoderma (Vanderwolf et al. 2013, Cunha et al. 2020, Zhang et al. 2021, Wasti et al. 2021); these genera were found in various substrates/hosts in caves (Raudabaugh et al. 2021, Martin-Pozas et al. 2022, Ogórek et al. 2022, Zalar et al. 2022). Three recent studies investigated cave fungi in China (Jiang et al. 2017, Zhang et al. 2017, 2021), and reported three, 20, and 33 novel species, respectively. In parallel investigations, such as those undertaken within Brazilian cave systems by Alves et al. (2022), Pseudolecanicillium was delineated as a new genus within the Cordycipitaceae. Concurrently, six new species were discovered from sediments and atmospheric samples: Aspergillus lebretii, Malbranchea cavernosa, Pseudohumicola cecavii, Pseudolecanicillium caatingaense, Talaromyces cavernicola, and Tritirachium brasiliense. Additionally, through morphological and multi-locus phylogenetic examinations, fungi were identified in association with bat flies in a Caatinga dry forest cave in Brazil. This analysis resulted in the description of two new species: Allophoma brasiliensis and Pyrenochaetopsis cecavii as reported by Carvalho et al. (2022). A number of studies have explored the diversity of fungi found in caves during 2020–2022; the results of these studies indicated high species diversity and demonstrated the presence of many new fungal taxa obtained from several hosts/substrates in caves in various parts of the world (Karunarathna et al. 2020, Takashima et al. 2020, Crous et al. 2020, Liu et al. 2021, Leplat et al. 2021, 2022, Pereira et al. 2022, Visagie et al. 2022). The description of these new species highlights the need for further research and documentation of the mycobiota that live in this environment. This study adds to our knowledge of the fungi that exist in harsh cave environments and highlights the importance of sustainable conservation and exploitation of these resources. Future research, including the integration of omics technologies (metagenomics) and culture-independent techniques, has the potential to deepen our understanding of the ecology of these fungi and their ecological functions within karst caves. This knowledge will be essential in identifying how cave microbiomes respond to increased human presence, establishing effective management practices, and implementing methods for restoring cave microbiomes affected by tourism in the Satun UNESCO Global Geopark.

Conflict of interest

The authors declare that there is no conflict of interest.

Supplementary information

Table S1.

Morphological comparisons of the new species (Actinomortierella caverna) with its close relatives.

Structure	A. caverna	A. ambigua	A. capitata	A. wolfii
Vegetative hypha	1.10–4.59 μm	-	-	-
Sporangiophores	79.9–353.0 × 6.4–18.2 μm	20.0–500.0 × 7.0–27.0 (base); 2.0–5.0 (apex) μm	350.0–500.0 × 18.0–23.0 μm	90.4–409.0 ×11.0–20.0 μm
Sporangium size	13.0–37.6 μm	4.4–17.0 μm	57.0–92.0 μm	11.3–67.8 μm
Sporangiospore shape	globose to subglobose	oblong	spherical	ellipsoid-reniform
Sporangiospore size	5.9–12.6 μm × 5.8–11.8 μm	4.0–9.0 × 3.0–6.0 μm	8.5–10.0 μm	3.3–13.2 × 2.2–5.5 μm
Vesicles	56.0–60.1 μm	4.4–17.0 μm	57.0–92.0 μm	3.3–5.5 μm
Branches	7.6–60.0 × 2.2–4.8 μm	4.0–18.0 μm long	8.0–14.0 × 2.5–4.0 μm	6.6–169.5 μm long
Chlamydospores	not observed	11.0–18.0	-	-
References	This study	Mehrotra et al. (1963)	Marchal (1891)	Mehrotra & Baijal (1963)

Table S2.

Morphological comparison of the new species (Leptobacillium latisporum) with its close relatives.

Fungus	Phialide size	Conidial size	Conidial shape and conidial chain or conidial head characteristics	Sources/habitats	References
L. cavernicola	5.1–27.2 × 1.2–1.7 μm	3.1–6.9 × 0.9–1.5 μm	Narrowly cylindrical to slightly fusoid, with some lemon-shaped (in long, slender chains)	Sample from cave	Leplat et al. (2022)
L. chinense	(6.0–) 15.0–30.0 (–68.0) × 1.5 μm	3.5–5.0 × 0.8–1.5 (–2.0) μm (av.: 4.2 × 1.3)	Ovoid, ellipsoidal, cylindrical	Coculture from isolation of freshwater fungi	Liu & Cai (2012)
L. coffeanum	11.0–44.0 (–70.0) × 1.0–2.4 μm	Macroconidia, 5.3–8.8 × 1.0–1.6 μm (av.: 6.1 × 1.3)	Spindle-shaped, ellipsoidal to fusoid, slightly curved (in subglobose to ellipsoidal heads)	Endophyte from Coffea arabica	Gomes et al. (2018)
		Microconidia, 2.2–3.8 × 0.8–1.5 μm (av.: 2.9 × 1.1)
L. filiforme	9.0–18.0 × 1.0 μm	7.2–12.5 × 1 μm	Fusoid to filiform (in long, sometimes zigzag, chains)	Endophyte from leaves of Citrullus lanatus	Crous et al. (2018)
L. leptobactrum	20.0–45.0 × 1.0–2.0 μm (base), 0.5–0.7 μm (apex)	4.5–8.0 × 0.8–1.5 (–2.0) μm	Cylindrical to fusiform		Zare & Gams (2016)
L. muralicola	20.0–45.0 × 1.0–2.0 μm (base), 0.5–0.7 μm (apex) averaged: 32.0 × 1.8 μm (base) 0.6 μm (apex)	4.5–6.0 × 1.0–2.0 μm (av.: 5.5 × 1.6)	Narrowly cylindrical (rod-shaped) to slightly fusoid (in very long, slender, inconspicuous chains)	White mouldy spots on the mural surface	Sun et al. (2019)
L. symbioticum	7.1–30.6 × 1.6–3.5 (averaged: 14.7 × 2.7) μm	4.0–6.9 × 0.7–1.6 (av.: 5.1 × 1.1) μm	Fusoid to narrowly cylindrical (in long,	Sori of a rust fungus	Okane et al. (2020)
			slender and inconspicuous chains)
L. latisporum	13.2–40.8 × 2.9–4.8 μm	3.9–6.3 × 1.9–3.9 μm	Fusoid to narrowly cylindrical (in long chains)	Soil in cave	This study