Two Novel Species of Talaromyces Discovered in a Karst Cave in the Satun UNESCO Global Geopark of Southern Thailand

Karst caves are oligotrophic environments that appear to support a high diversity of fungi. Studies of fungi in Thailand’s caves are limited. During a 2019 exploration of the mycobiota associated with soil samples from a karst cave, namely, Phu Pha Phet in the Satun UNESCO Global Geopark in Satun Province, southern Thailand, two previously undescribed fungi belonging to Talaromyces (Trichocomaceae, Eurotiales, Eurotiomycetes) were studied using a polyphasic approach combining phenotypic and molecular data. Based on datasets of four loci (ITS, BenA, CaM, and RPB2), phylogenetic trees of the section Trachyspermi were constructed, and two new species—Talaromyces phuphaphetensis sp. nov. and T. satunensis sp. nov.—phylogenetically related to T. subericola, T. resinae, and T. brasiliensis, are described. Detailed descriptions and illustrations of the new species are provided. This study increases the number of cave-dwelling soil fungi discovered in Thailand’s Satun UNESCO Global Geopark, which appears to be a unique environment with a high potential for discovering fungal species previously undescribed.


Introduction
The genus Talaromyces was introduced [1] with Talaromyces vermiculatus (=T. flavus) as the type of species. Talaromyces taxa are classified into Aspergillaceae, Eurotiales, Eurotiomycetidae, Eurotiomycetes, Pezizomycotina, and Ascomycota (MycoBank. 2022; Species Fungorum. 2022; accessed on 1 June 2022). This genus is well-known and among the most prevalent groups of fungi, found in a range of habitats, including soil, vegetation, air, living or decaying plants, indoor environments, and a wide range of food products [2][3][4][5][6][7]. Phu Pha Phet Cave, a part of a mycological diversity project associated with Satun Geopark, Thailand's first UNESCO Global Geopark, is also known as "Diamond Mountain Cave". It is the fourth largest cavern on earth and the largest cave in Thailand, covering more than 80,000 m 2 . Based on estimated visitation, the cave has been opened as a tourist attraction and is regarded as an anthropogenic disturbance; nonetheless, some areas in the Phu Pha Phet Cave remain closed [8]. Research on fungal diversity and mycological systematics in karst caves has been scarce in Thailand's Satun UNESCO Global Geopark.
In this study, soil samples randomly obtained from the Phu Pha Phet Cave were subjected to phenotypic examination and phylogenetic approaches, and two new cavedwelling soil micro-fungi belonging to Talaromyces-T. phuphaphetensis and T. satunensis spp. nov.-were described and compared with similar taxa.

Collection, Isolation, and Morphology
On 3 December 2019, collections were performed during a fungal survey of Phu Pha Phet Cave. Two strains of Talaromyces were isolated from soil samples (110 m elevation; 7 • 07 35 N 99 • 59 49 E) in Thungwa, Manang District, La-Ngu, Satun Province, southern Thailand. Ten or twenty grams of soil were randomly collected at shallow depths (1-5 cm) after removing the surface layer, placed in zip lock bags, preserved at 4 • C in an ice box during collection, and transferred to the mycological laboratory at the National Center for Genetic Engineering and Biotechnology (BIOTEC).
The dilution plate technique was carried out using a modified version of the method of Zhang et al. [9], and 1 g of the sample was suspended in 9 mL of sterile distilled water and then serially diluted 10-fold. Dilutions from 10 −1 to 10 −5 were prepared, and 100 µL of each dilution was spread on potato dextrose agar (PDA; Difco, GA, USA) containing two antibiotics (50 µg/mL of ampicillin and 50 µg/mL of streptomycin) with three replicates. Plate cultures were incubated at room temperature for two-three days to allow fungal growth before subculture onto PDA without antibiotics for additional morphological investigation.
After seven days, macroscopic features and growth rates were examined on seven traditional culture media (Czapek yeast autolysate agar (CYA), Czapek's agar (CZ), malt extract agar (MEA), yeast extract sucrose agar (YES), dichloran 18% glycerol agar (DG18), creatine sucrose agar (CREA), and oatmeal agar (OA, Difco)), as previously described [10]. Strains were inoculated with spore suspensions at three points and incubated in the dark at 25 • C, with additional temperatures of 30 and 37 • C for CYA. Extended incubation of MEA and OA plates for four weeks was performed to observe sexual reproduction. Microscopic observations were carried out on 7-day-old MEA, CZ, and CYA media. Ethanol (70%) and lactic acid (60%) were used to wash excess of conidia and mount slides, respectively.
Microscopic characters (i.e., conidiophores, conidiogenous cells, and conidia) were examined with a light microscope (OLYMPUS CX31; Olympus Corporation, Japan) and photographed using a Nomarski differential interference contrast microscope (OLYMPUS DP70). The Methuen Handbook of Color created color codes that were used to categorize the observed colors of the colonies [11]. The types and strains were deposited into the

DNA Extraction, PCR Amplification, and Phylogenetic Analyses
Following the protocols of Sri-indrasutdhi et al. [12], genomic DNA was extracted from 7-day-old cultures grown on MEA using the cetyltrimethylammonium bromide (CTAB) method. The internal transcribed spacer (ITS) region, β-tubulin (BenA), calmodulin (CaM), and RNA polymerase II (RPB2) genes were amplified. The primers and amplification profiles used are shown in Table 1. PCR products were purified and sequenced by Macrogen Inc. (Seoul, South Korea) using the same PCR primers used for PCR amplification. The obtained sequences of ITS, BenA, CaM, and RPB2 were assembled and trimmed at both ends in BioEdit v.7.1.3 [13]. The newly generated sequences were deposited in GenBank (the National Centre for Biotechnology Information (NCBI)), and representative Talaromyces in the section Trachyspermi used in phylogenetic analyses, and their accession numbers are provided in Table 2.

Phylogenetic Analysis
The phylogenetic trees of ITS, BenA, CaM, and RPB2 constructed separately using ML analyses and the concatenated datasets of four loci based on ML and Bayesian analyses revealed the relationships among the novel strains (TBRC 16281 and TBRC 16246) and Talaromyces species of the section Trachyspermi (Figures 1-3). Based on the single-gene analyses, our two proposed new species, T. phuphaphetensis and T. satunensis, were clustered with T. brasiliensis URM 7618, T. resinae CBS 324.83, and T. subericola CBS 144322. The two new species and T. subericola formed a monophyletic group, and were revealed as phylogenetically related to T. brasiliensis and T. resinae (Figures 1 and 2).
In the ITS and CaM phylograms, T. subericola was a sister taxon to T. phuphaphetensis, and these two lineages were closely related to T. satunensis with a good bootstrap support (Figures 1 and 2). In the BenA phylogram, our two new species clustered together with a low support value (bootstrap value < 70%) and were closely related to T. subericola on a highly supported branch (99%). In the RPB2 analyses (no sequence data of T. satunensis), T. subericola was the closest sister taxon to T. phuphaphetensis, with good bootstrap support (90%).
Based on the combined datasets of ITS, BenA, CaM, and RPB2, the phylogenetic relationships showed a topology similar to those obtained from each gene individually ( Figure 3). The two new species, T. phuphaphetensis and T. satunensis, formed two single branches and a well-supported clade with T. brasiliensis, T. resinae, and T. subericola (BS/PP = 95%/1.00). Talaromyces subericola was a sister taxon of T. phuphaphetensis, and these two species were closely related to T. satunensis in both fully supported subclades (BS/PP = 100%/1.00). Phylogenetically, T. resinae and T. brasiliensis were at a basal position, located on a single branch within the same clade as T. phuphaphetensis and T. satunensis.

Phylogenetic Analysis
The phylogenetic trees of ITS, BenA, CaM, and RPB2 constructed separately using ML analyses and the concatenated datasets of four loci based on ML and Bayesian analyses revealed the relationships among the novel strains (TBRC 16281 and TBRC 16246) and Talaromyces species of the section Trachyspermi (Figures 1-3). Based on the single-gene analyses, our two proposed new species, T. phuphaphetensis and T. satunensis, were clustered with T. brasiliensis URM 7618, T. resinae CBS 324.83, and T. subericola CBS 144322. The two new species and T. subericola formed a monophyletic group, and were revealed as phylogenetically related to T. brasiliensis and T. resinae (Figures 1 and 2).    In the ITS and CaM phylograms, T. subericola was a sister taxon to T. phuphaphetensis, and these two lineages were closely related to T. satunensis with a good bootstrap support (Figures 1 and 2). In the BenA phylogram, our two new species clustered together with a low support value (bootstrap value < 70%) and were closely related to T. subericola on a highly supported branch (99%). In the RPB2 analyses (no sequence data of T. satunensis), T. subericola was the closest sister taxon to T. phuphaphetensis, with good bootstrap support
100%/1.00). Phylogenetically, T. resinae and T. brasiliensis were at a basal position, located on a single branch within the same clade as T. phuphaphetensis and T. satunensis.
In addition, T. phuphaphetensis showed poor growth on CYA incubated at 37 • C (3-4 mm, 7 days), while T. brasiliensis, T. satunensis, and T. subericola had no growth on the medium. Morphological comparisons of T. phuphaphetensis, T. satunensis, and the three related species are shown in Table 3.
Note: Phylogenetically, T. satunensis is located within a terminal clade, and it is closely related to T. phuphaphetensis and T. subericola (Figure 3). Talaromyces subericola differs from our two new species in producing smooth-walled stipes and verruculose conidia. In comparison, T. satunensis differs from T. phuphaphetensis in the absence of diffusible pigments on CYA, lacking growth on CYA at 37 • C, and poor sporulation on MEA. In addition, T. satunensis has longer stipes and sometimes produces terverticillate branches (see Table 3).

Discussion
In this study, phylogenies and morphological characters supported the establishment of Talaromyces phuphaphetensis and T. satunensis as two new species belonging to Talaromyces section Trachyspermi. Phylogenetic analyses based on single loci (i.e., ITS, BenA, CaM, and RPB2) and the multi-locus approach showed that T. phuphaphetensis and T. satunensis are members of the Talaromyces clade composed of T. brasiliensis, T. resinae, and T. subericola. All phylogenetic trees also indicated that T. subericola has the closest relationship with our two new species described herein. Based on the combined dataset, the phylogenetic analyses revealed that T. brasiliensis and T. resinae are basal to T. phuphaphetensis, T. satunensis, and T. subericola (Figure 3).
The topology of the CaM tree for T. brasiliensis and T. resinae showed a slightly different position ( Figure 2). In addition, the species relationships within the section Tra-chyspermi, as shown in the phylogenetic trees inferred from CaM, were different from those in the trees based on the ITS, BenA, and RPB2 genes. These data are congruent with the studies of Rajeshkumar et al. [5] and Zhang et al. [22]. However, the phylogenetic tree of CaM gene sequences could distinguish T. phuphaphetensis and T. satunensis from other species in the section. Although the RPB2 gene is formally accepted as a potential molecular locus for identifying Talaromyces species, it is often difficult to amplify the targeted DNA region [23][24][25]. Unfortunately, this study did not obtain RPB2 sequence data from T. satunensis, although we attempted with different PCR profiles. Nonetheless, the phylogenies based on single genes and the concatenated data also confirmed the taxonomic placements of T. phuphaphetensis and T. satunensis as two distinct species in the Trachyspermi section.
Both T. phuphaphetensis and T. satunensis are characterized by the production of biverticillate conidiophores, tuberculate-walled stipes, and smooth-walled conidia. They grow restrictedly on CYA, YES, and DG18, slightly faster on MEA, and poorly on CREA. These data are in agreement with the description of the section [25,26]. Colonies of T. phuphaphetensis produce yellow pigment on CYA, CZ, and DG18. Generally, Talaromyces species are reported to be good pigment producers [27][28][29]. Many species in the section Trachyspermi (such as T. albobiverticillius, T. atroroseus, and T. minioluteus) can produce a large amount of red pigment. However, only T. atroroseus produces pigments without known mycotoxins, which might be suitable for application in the food or healthcare industry as an alternative synthetic dye [27]. Likewise, the new species we propose can serve as an alternative source of natural pigments that need to be investigated for mycotoxin production, enhanced pigment production, and other testing for future research.

Conclusions
Two isolates of soil fungi were discovered in the Phu Pha Phet Cave of the Satun UN-ESCO Global Geopark in southern Thailand and identified as part of the genus Talaromyces in the section Trachyspermi. The two isolates are proposed as new species, namely Talaromyces phuphaphetensis and T. satunensis, based on their morphological and phylogenetic differences from the other species described in the section Trachyspermi. The discovery will support future evaluations of the unique species' potential applications and functions. Information on the mycological biodiversity and habitat of UNESCO's Satun cave would promote awareness of sustainable conservation and exploitation, supporting the future planning, monitoring, and management of Thai caves in achieving a balance between conservation and development. Furthermore, the results contribute to the knowledge of cave-dwelling soil fungi, their ecological uniqueness and diversity in Thailand, and their global geographical distribution. Interestingly, it is also possible that more new species will be discovered in this peculiar environment in Thailand's Satun UNESCO Global Geopark.