Discovery and Extrolite Production of Three New Species of Talaromyces Belonging to Sections Helici and Purpurei from Freshwater in Korea

Three novel fungal species, Talaromyces gwangjuensis, T. koreana, and T. teleomorpha were found in Korea during an investigation of fungi in freshwater. The new species are described here using morphological characters, a multi-gene phylogenetic analysis of the ITS, BenA, CaM, RPB2 regions, and extrolite data. Talaromyces gwangjuensis is characterized by restricted growth on CYA, YES, monoverticillate and biverticillate conidiophores, and globose smooth-walled conidia. Talaromyces koreana is characterized by fast growth on MEA, biverticillate conidiophores, or sometimes with additional branches and the production of acid on CREA. Talaromyces teleomorpha is characterized by producing creamish-white or yellow ascomata on OA and MEA, restricted growth on CREA, and no asexual morph observed in the culture. A phylogenetic analysis of the ITS, BenA, CaM, and RPB2 sequences showed that the three new taxa form distinct monophyletic clades. Detailed descriptions, illustrations, and phylogenetic trees are provided.


Introduction
The genus Talaromyces was established by Benjamin (1955) [1] for a teleomorph of Penicillium with Talaromyces vermiculatus (=T. flavus) as the type species. These species are characterized by cleistothecial or gymnothecial ascomata, unitunicate eight-spored asci, and unicellular ascospores with or without equatorial crests. The anamorphs have predominantly biverticillate or rarely terverticillate conidiophores with acerose phialides and narrow collulum [2,3]. In 2011, Samson et al. [2] transferred all accepted species of Penicillium subgen. Biverticillium to Talaromyces on the basis of a two-gene phylogeny. Subsequently, Yilmaz et al. [3] studied the taxonomy of Talaromyces in detail using the polyphasic species concept. On the basis of multigene phylogeny, morphology, and physiology, Yilmaz et al. [3] placed 88 accepted species in seven well-defined sections, namely, Bacillispori, Helici, Islandici, Purpurei, Subinflati, Talaromyces, and Trachyspermi. However, the lists are rapidly increasing with many new Talaromyces species recently described from all over the world and added to sections Helici, Islandici, Purpurei, Subinflati, Talaromyces, and Trachyspermi . To date, 171 species have been reported in the genus Talaromyces [27], of which only three species: Talaromyces angelicae, Talaromyces cnidii, and Talaromyces halophytorum were reported from Korea [28,29]. Recently, a new section Tenues was proposed [26]. Talaromyces contains species that play an important role in agriculture and biotechnology. Talaromyces rugulosus (Basionym: Penicillum rugulosum) produces β-rutinosidase and phosphatase [30,31], T. pinophilus (Basionym: Penicillium pinophilum) produces endoglucanase and cellulase [32], and T. funiculosus (Basionym: Penicillium funiculosum) produces cellulases [33]. Talaromyces purpureogenus can produce extracellular enzymes and red pigment and also produces mycotoxin such as rubratoxin A and B and luteoskyrin [34]. Additionally, red pigments produced in large amounts by T. atroroseus can be used as colorants in the food industry [35]. Furthermore, the ability to produce various important compounds makes them candidates for the biocontrol of soilborne fungal pathogens such as an antagonists of T. flavus against Verticillium spp., Rhizoctonia solani, and Sclerotinia sclerotiorum [36][37][38][39][40]. In addition, some species are medically important, such as T. wortmannii, which can produce compound C that was found to be an effective antimicrobial against Propionibacterium acnes and had anti-inflammatory properties and, thus, represents alternative treatments for antibiotic or anti-inflammatory therapy for acne [41]. Talaromyces marneffei (Basionym: Penicillium marneffei) causes a fatal mycosis in immunocompromised individuals [42,43].
Section Helici was proposed by Yilmaz et al. [3] with seven Talaromyces species divided into two clades: a main clade containing T. helicus, T. boninensis, and T. varians and a second clade containing T. cinnabarinus, T. aerugineus, T. bohemicus, and T. ryukyuensis. The Talaromyces species included in this section are characterized by producing biverticillate conidiophores occasionally consisting of solitary phialides with stipes generally pigmented, yellowish-brown, or dark green reversed on CYA; grown at 37 • C, and the absence of acid production on CREA [3]. Section Helici currently includes 13 species [27].
Section Purpurei was proposed by Stolk and Samson [44] to accommodate species that produce synnemata after two to three weeks of incubation, with the exception of T. rademirici, T. purpureus, and T. ptychoconidium. The species in this section generally do not grow or grow poorly on creatine sucrose agar (CREA), and grow restrictedly on Czapek yeast extract agar (CYA) and yeast extract sucrose agar (YES) and slightly faster on malt extract agar (MEA) [3]. Ten species were accepted in the section Purpurei: T. cecidicola, T. chloroloma, T. coalescens, T. dendriticus, T. pseudostromaticus, T. pittii, T. purpureus, T. ptychoconidium, T. rademirici, and T. ramulosus [3], but it currently contains 12 species [27].
Freshwater fungi are an ubiquitous and diverse group of organisms and play an important role in ecological systems [45]. Hawksworth [46] estimated that there are approximately 1.5 million fungal species on Earth. However, an updated estimate of the number of fungal species is between 2.2 and 3.8 million [47]. Of the ca. 150,000 known sepecies, only around 3000 have been reported from aquatic habitats [48], with more than 600 species of ascomycetes reported in freshwater [49]. Thus, a large number of species are still waiting to be discovered and described in freshwater habitats.
Up to now, only a few freshwater fungi, especially genus Talaromyces, have been reported in Korea. The purpose of this study was to expand the present knowledge of these fungal taxa in Korea. Here, we describe and illustrate three new Talaromyces species from freshwater habitats in Korea.

Sampling and Isolation
In January and May 2017, freshwater samples were collected from the Wonhyo Valley located at Mudeung Mt., Gwangju, and Jukrim Reservoir located in Yeosu, Korea. These samples were transported to the laboratory in sterile 50-mL conical tubes and stored at 4 • C pending examination. Before culture preparation, all samples were diluted with sterile distilled water to reduce the density and improve strain recovery. Briefly, each sample was shaken for 15 min at room temperature, and a 100-µL aliquot of each sample was mixed with 9 mL of sterile distilled water. Then, serial dilutions of the mixture (from 10 −1 to 10 −4 ) were made. A 100-µL aliquot of each dilution was spread on potato dextrose agar (PDA: 39 g of potato dextrose agar in 1 L of deionized water; Becton, Dickinson, and Co., Sparks, MD, USA) supplemented with the antibiotic streptomycin (final concentration, 50 ppm; Sigma-Aldrich, St. Louis, MO, USA). The petri plates were incubated at 25 • C for 5-10 days. Pure isolates were obtained by selecting individual colonies of varied morphologies, transferring them to PDA plates, and subculturing until pure cultures were obtained. Ex-type living cultures were deposited in the Environmental Microbiology Laboratory Fungarium, Chonnam National University (CNUFC), Gwangju, Korea. Dried cultures were deposited in the Herbarium Chonnam National University, Gwangju, Korea.

DNA Extraction, PCR, and Sequencing
The fungal isolates were cultured on PDA overlaid with cellophane at 25 • C for 5-7 days. Genomic DNA was extracted using the Solg TM Genomic DNA Preparation Kit (Solgent Co. Ltd., Daejeon, Korea). The ITS region was amplified using the primer pairs ITS 1 and ITS 4 [51]. The beta-tubulin (BenA) was amplified using the primer pairs T10 and Bt2b [52]. The calmodulin (CaM) gene was amplified using the primer pairs CMD5/CMD6 and CF1/CF4 [53,54]. To amplify the RPB2 gene region, the primer pairs RPB2-5F and RPB2-7cR were used [55]. PCR amplification was performed according to the conditions described by Yilmaz et al. [3] and Houbraken and Samson [56]. The PCR products were purified with the Accuprep PCR Purification Kit (Bioneer Corp., Daejeon, Korea). Sequencing was performed using the same PCR primers and run on the ABI PRISM 3730XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).

Molecular Analysis
Each generated sequence was checked for the presence of ambiguous bases and assembled using the Lasergene SeqMan program from DNASTAR, Inc. (Madison, WI, USA). Edited sequences were blasted against the NCBI GenBank nucleotide database (https://blast.ncbi.nlm.nih.gov/Blast.cgi; 2 January 2021) to search for the closest relatives. The sequences of all the accepted Talaromyces species were retrieved from GenBank. The sequences were aligned using MAFFT (https://mafft.cbrc.jp/alignment/server; 9 March 2021) [57], and the resulting alignment was trimmed using trimAl [58] and subsequently combined with MEGA 7 [59]. The data were converted from a FASTA format to nexus and phylip formats using the online tool Alignment Transformation Environment (https://sing.ei.uvigo.es/ALTER/; 9 March 2021) [60]. Phylogenetic reconstructions by maximum likelihood (ML) were carried out using RAxML-HPC2 on XSEDE on the online CIPRES Portal (https://www.phylo.org/portal2; 9 March 2021) with 1000 bootstrap replicates and the GTRGAMMA model of nucleotide substitution. A Bayesian inference analysis was performed with MrBayes 3.2.2 [61] using a Markov Chain Monte Carlo (MCMC) algorithm. The sample frequency was set to 100, and the first 25% of trees were removed as burn-in. The trees were visualized using FigTree v. 1.3.1 [62]. Support values were provided at the branches (ML bootstrap support (BS) and BI posterior probability (PP)). Talaromyces tenuis CBS 141840 was chosen as the outgroup in the sections Helici and Purpurei phylogenies. Trichocoma paradoxa CBS 788.83 was the outgroup for the combined phylogeny of the species from Talaromyces. The newly obtained sequences were deposited in the GenBank database under the accession numbers provided in Table 1.

Extrolite Analysis
Extrolites were extracted from Talaromyces strains after growing on CYA, YES, and MEA for 7-10 days at 25 • C. The extracts were prepared and analyzed as previously described by Frisvad and Thrane [63], Nielsen et al. [64], and Houbraken et al. [65].

Phylogenetic Analysis
Phylogenetic relationships within Talaromyces were studied using a concatenated dataset of four loci (ITS, BenA, CaM, and RPB2) (Figure 1). The multigene analysis contained 67 taxa, including Trichocoma paradoxa CBS 788.83 as the outgroup taxon. The concatenated alignment consisted of 2407 characters (including alignment gaps): 425, 443, 687, and 852 characters used in the ITS, BenA, CaM, and RPB2, respectively. Eight main lineages are present within Talaromyces, which agrees with the sectional classification by Yilmaz et al. [3] and Sun et al. [26]. In the phylogenetic analysis, a small clade containing T. brunneosporus highlighted by asterisk could not be assigned to any known sections ( Figure 1). Talaromyces gwangjuensis, T. koreana, and T. teleomorpha belong to sections Purpurei and Helici, according to our multigene analysis (Figure 1). In section Purpurei, T. gwangjuensis clustered close to but separated from T. rademirici in the single (BenA, RPB2, and ITS) and combined phylogenies (Figure 2 and Figures S1-S3). Talaromyces teleomorpha is close to T. helicus in BenA, ITS, and combined phylogenies (Figure 3, Figures S4 and S5) but placed among T. helicus, T. koreana, T. reverso-olivaceus, and T. boninensis in the CaM and RPB2 phylogenies ( Figures S6 and S7). Talaromyces koreana was found to be related to T. reverso-olivaceus and T. boninensis in BenA, CaM, RPB2, and the combined phylogenies ( Figure 3, Figures S4,  S6, and S7). In the ITS phylogenetic analysis, T. koreana was close to only T. boninensis ( Figure S5).
Extrolites: Cycloleucomelone, gregatin A, and purpactin A were detected in the ex-type strain of T. koreana.
Notes: Talaromyces koreana belongs to section Helici and is phylogenetically related to T. boninensis and T. reverso-olivaceus. Talaromyces koreana differs from T. boninensis and T. reverso-olivaceus by having a higher number of phialides per metula. Talaromyces koreana produces smaller conidia than those of T. boninensis and T. reverso-olivaceus.    Colony characters: CYA 25 • C, 7 d: Colonies raised at the center, slightly sulcate; margins low, plane, entire (3 mm); mycelia white to light yellow; reverse ivory to light yellow, slightly sunken at the center. MEA 25 • C, 7 d: colonies low, plane; mycelia white to light yellow, hyaline; reverse light orange at the center. YES 25 • C, 7 d: Colonies raised at the center, sulcate; margins low; mycelia white; reverse pale orange. OA 25 • C, 7 d: Colonies low, plane; mycelia white to light yellow, hyaline, smooth or rough, studded. CREA 25 • C, 7 d: Acid production absent.
Notes: Talaromyces teleomorpha can be distinguished easily from the closely related species T. helicus by growing rapidly on CYA, YES, and MEA at 25 • C in 7 days. Ascomata size of T. helicus are smaller than in T. teleomorpha. Talaromyces helicus does not grow on CREA, whereas T. teleomorpha can grow on this medium. In addition, T. teleomorpha does not produce the asexual morph, which is present in T. helicus.

Discussion
During a survey of fungi from a freshwater niche in Korea, three novel species were identified, namely Talaromyces gwangjuensis, T. koreana, and T. teleomorpha.
In our phylogenetic analysis, Talaromyces gwangjuensis was classified in section Purpurei. This species is closely related to T. rademirici, which also has both monoverticillate and biverticillate conidiophores and do not grow on CREA. However, Talaromyces gwangjuensis has more restricted colonies on YES and CYA and larger numbers of metulae and phialides. Growth on CYA at 37 • C and the conidial shape and size on MEA at 25 • C can be easily used to distinguish between T. gwangjuensis and T. rademirici. Talaromyces rademirici grows faster on CYA at all temperatures (CYA at 25 • C, 5-6; CYA at 30 • C, 5-7; CYA at 37 • C, 3), whereas Talaromyces gwangjuensis was unable to grow on CYA at 37 • C. Some species in this section have been reported to not grow on CYA at 37 • C, including T. pittii and T. purpureus [3]; however, T. pittii and T. purpureus produce ellipsoidal and subglobose to ellipsoidal conidia compared with T. gwangjuensis that produces globose conidia.
Talaromyces koreana and T. teleomorpha belong to the section Helici, which was established by Yilmaz et al. [3]. The species in the section was not found to produce acid on CREA medium [3]. However, recent studies showed that T. georgiensis and T. borbonicus could produce acid on the medium [12,20]. In the present study, T. koreana was also found to produce acid on the medium. The results suggest that the ability to produce acid on CREA may not usually a key character to distinguish this section. It is a common character for the species in the section Helici to be able to grow at 37 • C [3]. Our results are the same as previous studies [3]. Interestingly, we found that T. koreana could grow at 40 • C on MEA media (10-13 mm after 7 days), while not on other media. Our findings showed that the medium composition might influence the maximum growth of fungi.
Talaromyces teleomorpha is closely related to T. helicus. However, T. helicus produces both asexual and sexual morphs, whereas the asexual morph is not observed in T. teleomorpha [3]. Especially, T. teleomorpha can grow on CREA, while T. helicus is unable to grow on this medium [3].
Although ITS is the barcoding marker for fungi [66], this locus is not sufficient to differentiate all Talaromyces species. Yilmaz et al. [3] proposed using BenA as a secondary molecular marker. In this study, T. gwangjuensis, T. koreana, and T. teleomorpha could be separated via each single gene phylogram. Recently, T. brunneosporus was described as a new species discovered from honey in Spain [24]. It was assigned to section Purpurei using the ITS, BenA, CaM, and RPB2 concatenated dataset. The comparison of ITS, BenA, CaM, and RPB2 sequences deposited in GenBank indicated that this species could not be assigned to any known section based on our phylogenetic analyses (Figure 1). In each single gene phylogeny (ITS, BenA, CaM, and RPB2), T. brunneosporus also formed a separate lineage (data not shown). More strains are essential to confirm the taxonomic position of T. brunneosporus.
Some members from the genus Talaromyces are of great interest to the biotechnology industry in medial and food mycology because of their ability to produce a wide range of metabolites [3]. The species of section Purpurei produce various extrolite profiles. For example, T. cecidicola produces apiculides, pentacecilides, and thailandolides. Talaromyces coalescens, T. dendriticus, and T. purpurogenus share productions of penicillides, purpactins, and vermixocins. On the other hand, T. purpurogenus and T. pseudostromaticus produce the extrolite mitorubin. Some Talaromyces species produce mycotoxins such as botryodiplodin by T. coalescens, rugulovasine and luteoskyrin by T. purpurogenus, rubratoxins by T. purpurogenus and T. dendriticus, and secalonic acids D and F by T. pseudostromaticus. Talaromyces gwangjuensis, described in this study, produces austin, austinol, mitorubrin, mitorubrinol, mitorubrinol acetate, mitorubrinic acid, and a purpactin without any production of mycotoxins. Some secondary metabolites were found in the section Helici, such as alternariol, bacillisporin, and helicusins produced by T. helicus [3,67]. Talaromyces reverso-olivaceus produced rugulovasine A [5], while talaroderxines is produced by T. boninensis [3]. In this study, T. koreana produced cycloleucomelone, gregatin A, and purpactin A. Talaromyces teleomorpha also produced helicusins, as described by Yoshida et al. [67].
Talaromyces species are geographically distributed in many kinds of substrates. The species of section Helici have been reported to be isolated from soil, cotton yarn, debris, clinical sources, indoor environments, and biomass of Arundo donax [3,5,12,15,20]. The species of section Purpurei have been reported to be isolated from the air, wasp insect galls, Eucalyptus, Protea repens infructescence, and other substrates such as apples [3,17,[68][69][70][71]. In this study, we isolated three novel species from freshwater. As far as we know, only species belonging to section Talaromyces were reported from water [22,[72][73][74]. It is interesting to note that Talaromyces gwangjuensis, T. koreana, and T. teleomorpha were the first species in the sections Purpurei and Helici isolated from freshwater. Our studies expanded our knowledge on the substrates where Talaromyces species can occur. Further studies are needed for a better understanding of the ecological roles of these species.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/jof7090722/s1: Figure S1: Phylogram generated from the Maximum Likelihood (RAxML) analysis based on the BenA sequence data for species classified in Talaromyces section Purpurei. The branches with values = 100% ML BS and 1 PP are highlighted by thickened branches. The branches with values ≥ 70% ML BS and ≥ 0.95 PP indicated above or below branches. Talaromyces tenuis CBS 141840 was used as the outgroup. The newly generated sequences are indicated in blue. T = ex-type. Figure S2: Phylogram generated from the Maximum Likelihood (RAxML) analysis based on the RPB2 sequence data for species classified in Talaromyces section Purpurei. The branches with values = 100% ML BS and 1 PP are highlighted by thickened branches. The branches with values ≥ 70% ML BS and ≥ 0.95 PP indicated above or below branches. Talaromyces tenuis CBS 141840 was used as the outgroup. The newly generated sequences are indicated in blue. T = ex-type. Figure  S3: Phylogram generated from the Maximum Likelihood (RAxML) analysis based on the ITS sequence data for species classified in Talaromyces section Purpurei. The branches with values = 100% ML BS and 1 PP are highlighted by thickened branches. The branches with values ≥ 70% ML BS and ≥ 0.95 PP indicated above or below branches. Talaromyces tenuis CBS 141840 was used as the outgroup. The newly generated sequences are indicated in blue. T = ex-type. Figure S4: Phylogram generated from the Maximum Likelihood (RAxML) analysis based on the BenA sequences data for species classified in Talaromyces section Helici. The branches with values = 100% ML BS and 1 PP are highlighted by thickened branches. The branches with values ≥ 70% ML BS and ≥ 0.95 PP indicated above or below branches. Talaromyces tenuis CBS 141840 was used as the outgroup. The newly generated sequences are indicated in blue. T = ex-type. Figure S5: Phylogram generated from the Maximum Likelihood (RAxML) analysis based on the ITS sequences data for species classified in Talaromyces section Helici. The branches with values = 100% ML BS and 1 PP are highlighted by thickened branches. The branches with values ≥ 70% ML BS and ≥ 0.95 PP indicated above or below branches. Talaromyces tenuis CBS 141840 was used as the outgroup. The newly generated sequences are indicated in blue. T = ex-type. Figure S6: Phylogram generated from the Maximum Likelihood (RAxML) analysis based on the CaM sequence data for species classified in Talaromyces section Helici. The branches with values = 100% ML BS and 1 PP are highlighted by thickened branches. The branches with values ≥ 70% ML BS and ≥ 0.95 PP indicated above or below branches. Talaromyces tenuis CBS 141840 was used as the outgroup. The newly generated sequences are indicated in blue. T = ex-type. Figure S7: Phylogram generated from the Maximum Likelihood (RAxML) analysis based on the RPB2 sequence data for species classified in Talaromyces