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New Species and Eight Undescribed Species Belonging to the Families Aspergillaceae and Trichocomaceae in Korea

Abstract During a survey of fungal diversity associated with insects, mud, soil, and freshwater niches in different areas in Korea, nine interesting fungal strains were isolated. Based on their morphological characteristics and molecular phylogeny analyses, using a combined data set of β-tubulin (BenA), calmodulin (CaM), and second largest subunit of RNA polymerase (RPB2) sequences, the strains CNUFC AM-44, CNUFC JCW3-4, CNUFC S708, CNUFC WT202, CNUFC AS1-29, CNUFC JCW3-5, CNUFC JDP37, and CNUFC JDP62 were identified as Aspergillus alabamensis, A. floridensis, A. subversicolor, Penicillium flavigenum, P. laevigatum, P. lenticrescens, Talaromyces adpressus, and T. beijingensis, respectively. The strain CNUFC JT1301 belongs to series Westlingiorum in section Citrina and is phylogenetically related to P. manginii. However, slow growth when cultivated on CYA, MEA, CREA is observed and the property can be used to easily distinguish the new species from these species. Additionally, P. manginii is known to produce sclerotia, while CNUFC JT1301 strain does not. Herein, the new fungal species is proposed as P. aquadulcis sp. nov. Eight species, A. alabamensis, A. floridensis, A. subversicolor, P. flavigenum, P. laevigatum, P. lenticrescens, T. adpressus, and T. beijingensis, have not been previously reported in Korea. The present study expands the known distribution of fungal species belonging to the families Aspergillaceae and Trichocomaceae in Korea.


Introduction
Aspergillus, Penicillium, and Talaromyces are wellknown species of the Aspergillaceae and Trichocomaceae families, have a worldwide distribution, and have been isolated from a diverse range of habitats from air to soil, to vegetation and indoor environments [1][2][3]. Many species of Aspergillus, Penicillium, and Talaromyces are important both in industry and medicine, whereas others are associated with food spoilage, mycotoxin production, and dangerous human and plant diseases [4,5]. Various species can grow at low or high temperatures, low pH values, high salt or sugar concentrations, or low oxygen levels [6,7].
The genus Aspergillus was first introduced by Micheli in 1729 [8] to describe asexual fungi whose conidiophores resembled an aspergillum. Later, in 1768, Haller [9] validated the genus; in 1832, Fries [10] sanctioned the generic name, with Aspergillus glaucus (L.) Link [¼ Mucor glaucus L. Monilia glauca (L.) Pers.] as the generic type. Members of this genus produce many different compounds, which are used or have potential to be used in pharmacology and biotechnology [1,5]. Aspergillus is subdivided into 6 subgenera, 27 sections, and 75 series [11]. Currently, this genus consists of 446 species [11].
The genus Talaromyces was established by Benjamin (1955) [13] for a teleomorph of Penicillium, with Talaromyces vermiculatus (¼T. flavus) as the type species. These species are characterized by cleistothecial or gymnothecial ascomata, unitunicate 8-spored asci, and unicellular ascospores with or without equatorial crests. The anamorphs have predominantly biverticillate or rarely terverticillate conidiophores with acerose phialides and narrow column [3,14]. The genus is divided into seven sections based on multigene phylogenetic, morphological, and physiological characteristics reported by Yilmaz et al. [3]. Recently, a new section Tenues was proposed by Sun et al. [15]. Currently, the genus contains 171 accepted species [11]. Although the number of new species in the genera Aspergillus, Penicillium, and Talaromyces has increased in Korea, this number is significantly lower than the global number of accepted species. In addition, some habitats such as freshwater and insect hosts are still poorly studied.
The objective of this study was to morphologically and molecularly characterize of a new species, P. aquadulcis, and eight previously unrecorded fungal species in Korea-namely A. alabamensis, A. floridensis, A. subversicolor, P. flavigenum, P. laevigatum, P. lenticrescens, T. adpressus, and T. beijingensis-isolated from freshwater, insects, mud, and soil.

Sample collection and isolation
Freshwater, insect, mud and soil samples were collected from different areas in Korea. The samples were collected in sterile plastic bags or sterile 50 mL Falcon tubes and transferred to the laboratory. To isolate fungi from freshwater, soil, and mud, a serial dilution plating method was used. Briefly, 1 mL of water or 1 g of soil or mud was mixed with 9 mL of sterile distilled water; serial dilutions ranging from 10 À1 to 10 À5 were prepared. An aliquot of 0.1 mL from each dilution was transferred to potato dextrose agar (PDA; Difco, Becton, Dickinson and Co., Sparks, MD, USA) or yeast malt extract agar (YMA; Difco) supplemented with the antibiotic streptomycin sulfate (100 ppm) and was incubated at 25 C for 3-7 days. To isolate pure cultures, individual colonies with various morphologies were picked, transferred to PDA, and subcultured until pure mycelia were obtained. Fungal isolation from insect samples were conducted following our previous methods [16]. All

Morphological characteristics
The strains were three-point inoculated onto Czapek yeast autolysate agar (CYA; yeast extract, Difco), malt extract agar (MEA; Difco), yeast extract sucrose agar (YES; yeast extract, Difco), except creatinine sucrose agar (CREA) only for CNUFC JT1301 strain. All petri dishes were inoculated at 25 C for 7 days. Medium preparation, inoculation, and incubation were performed according to the methods reported by Yilmaz et al. [3]. Colony characteristics were recorded after 7 days. Lactic acid (60%) was used as the mount fluid, and 96% ethanol was used to remove excess conidia. The cultures were examined and photographed under an Olympus BX53 light microscope with an attached Olympus DP74 camera (Olympus, Tokyo, Japan). For SEM, samples were performed as described previously by Nguyen et al. [17]. The samples were then sputtercoated with gold and imaged with scanning electron microscope (Hitachi S4700 Field-Emission SEM, Hitachi, Tokyo, Japan) at the Korea Basic Science Institute, Gwangju, Korea.

DNA extraction, PCR, and sequencing
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 internal transcribed spacer (ITS) region was amplified using the primer pairs V9G and ITS4 [18,19]. b-tubulin (BenA) was amplified using the primer pairs T10 and Bt2b [20]. Calmodulin (CaM) was amplified using the primer pairs CF1 and CF4 [21,22]. Second largest subunit of RNA polymerase (RPB2) was amplified using the primer pairs RPB2-5F and RPB2-7cR [23]. PCR amplification was performed according to the conditions described by Visagie et al. [2] and Yilmaz et al. [3]. The PCR products were then purified using an Accuprep PCR Purification Kit (Bioneer Corp., Daejeon, Korea) and sequenced on the ABI PRISM 3730XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) using the same primers employed for PCR.

Phylogenetic analysis
Sequences for the selected strains were retrieved from the GenBank and aligned using MAFFT (http://mafft. cbrc.jp/alignment/server) with the algorithm L-INS-I. Aligned sequences were automatically trimmed using trimAl [24] using the gappyout method. Concatenated datasets of the BenA, CaM and RPB2 genetic markers were built using MEGA7 [25]. Phylogenetic reconstructions by maximum likelihood (ML) was carried out using RAxML-HPC2 on XSEDE on the online CIPRES Portal (https://www.phylo.org/portal2) with bootstrap

Molecular phylogeny
Combined BenA, CaM and RPB2 was used to clarify the phylogenetic relationships of newly collected specimens with other species in the genera Aspergillus, Penicillium, and Talaromyces (Figures 1-4). The three-locus phylogenetic tree with the 41 isolates of Aspergillus is shown in Figure 1. The concatenated alignment consists of 2,227 characters (including alignment gaps): 541, 676, and 1,010 characters used in the BenA, CaM, and RPB2, respectively. The isolates CNUFC S708, CNUFC JCW3-4, and CNUFC AM-44 are clustered within the same clade as Aspergillus subversicolor CBS 145751 (ex-type strain), A. floridensis NRRL 62478 (ex-type strain), and A. alabamensis CBS 125693 (ex-type strain), in the sections Nidulantes, Nigri, and Terrei, respectively.
The phylogenetic trees generated by the concatenated BenA-CaM-RPB2 sequences (424, 528, 915 and 1,867 characters with gaps, respectively) indicates that our two isolates CNUFC JT1301 and CNUFC JT1302 are related to P. manginii with high statistical support ( Figure 2).
The phylogenetic tree based on the three-locus with the 45 isolates of Penicillium is shown in Figure 3. The concatenated alignment consists of    Colony characteristics: On CYA, colonies floccose, plane; mycelium white; sporulation moderate; soluble pigments absent; reverse pale yellowish brown; and reached 28-30 mm in diameter after 7 days at 25 C. On MEA, colonies floccose, plane; mycelium white to yellowish brown at the center; sporulation moderate to dense; soluble pigments absent; reverse pale-yellow brown; and reached 33-35 mm in diameter after 7 days at 25 C. On YES, colonies plane, slightly sulcate; mycelium white; sporulation in the colony center; soluble pigments absent; reverse pale orange; and reached 31-33 mm in diameter after 7 days at 25 C.
Note: Our phylogenetic analyses showed that P. aquadulcis forms a separate lineage, phylogenetically related to P. manginii. However, slow growth when cultivated on CYA, MEA, CREA is observed and the property can be used to easily distinguish the new species from these species. The conidial shape of P. aquadulcis (globose to subglobose) is different from that of P. manginii (ellipsoidal). Additionally, P. manginii is known to produce sclerotia, while P. aquadulcis does not.  Note: There were differences observed in the colony diameter for CNUFC WT202 in comparison to previous descriptions. Its colony diameter was different from that of previously described Penicillium flavigenum on CYA (21-31 mm) [31]. In this isolate, conidia were slightly smaller than Penicillium flavigenum (2.5-4 mm) [31]. Colony characteristics: On CYA, colonies slightly sulcate; margins entire; mycelium white; sporulation sparse; soluble pigments absent; exudates absent; reverse light grayish yellow; and reached 21-23 mm in diameter after 7 days at 25 C. On MEA, mycelium white; slightly raised at the center; sporulation strong; soluble pigments absent; exudates absent; reverse light orange yellow; and reached 36-40 mm in diameter after 7 days at 25 C. On YES, colonies slightly raised at the center; margins entire; mycelium white; sporulation sparse; soluble pigments absent; exudates absent; reverse buff-yellow; and reached 23-25 mm in diameter after 7 days at 25 C. Note: Based on phylogenetic analysis of combined BenA, CaM, and RPB2 sequence data, our isolate CNUFC AS1-29 was clustered with the ex-type strains of P. laevigatum and P. wandoense in laevigatum clade (Figure 3). The colony morphology and culture characteristics of CNUFC AS1-29 strain were similar to the previous descriptions [32]. As reported by Houbraken et al. [11], P. laevigatum and P. wandoense, which were isolated from freshwater collected at Wando, Korea [33], were conspecific. However, P. wandoense (3-5 mm) differs from P. laevigatum (1-14 mm) by having smaller number of phialides per metulae [32]. Penicillium wandoense also differs from P. laevigatum (type species) and isolate CNUFC AS1-29 in terms of reverse color on MEA. Thus, future morphology and molecular studies are needed to confirm the relationship between P. laevigatum and P. wandoense.  (Figure 11).
Colony characteristics: On CYA, colonies sulcate; margins entire; mycelia white; sporulation moderate; soluble pigments absent; exudates absent; reverse pale yellow; and reached 23-29 mm in diameter after 7 days at 25 C. On MEA, colonies slightly raised at the center, plane; mycelium white; soluble pigments absent; exudates absent; reverse yellowish gray; and reached Note: The morphological characteristics of the isolate were consistent with those of P. lenticrescens, as described by Visagie et al. [2]. However, our strain produced biverticillate and divaricate conidiophores, in contrast to the mostly biverticillate sporangiophores of P. lenticrescens described by Visagie et al. [2]. Colony characteristics: On CYA, colonies slightly sulcate; margins entire; mycelium white; texture floccose; sporulation moderately to dense; soluble pigments absent; exudates absent; reverse yellowish orange, and reached 22-24 mm in diameter after 7 days at 25 C. On MEA, colonies slightly raised at the center, plane; mycelium white; sporulation sparse; texture floccose in the center; soluble pigments absent; exudates absent; reverse light yellow; and reached 32-34 mm in diameter after 7 days at 25 C. On YES, colonies raised at the center; appearance green; margins entire; mycelium white; Note: The morphological characteristics of the isolate CNUFC JDP37 in this study were similar to those of the previously described T. adpressus by Chen et al. [34]. However, our observations showed that the size of conidia of the isolate CNUFC JDP37 was smaller than that of previously reported species. Colony characteristics: On CYA, colonies moderately deep, raised at the center; margins entire; mycelium white; texture velvety; sporulation dense; soluble pigments absent; reverse peach; and reached 26-29 mm in diameter after 7 days at 25 C. On MEA, colonies slightly raised at the center, plane; mycelium white; sporulation sparse; reverse light yellow; and reached 31-33 mm in diameter after 7 days at 25 C. On YES, colonies raised at the center; appearance green; margins entire; mycelium white; sporulation moderate; soluble pigments absent; exudates absent; reverse brownish orange at the center, yellow at the edge; and reached 29-31 mm in diameter after 7 days at 25 C.
Micromorphology: Conidiophores mostly terverticillate, sometimes biverticillate, stipes smooth, 2.5-3.5 mm.  Note: The morphological characteristics of the isolate CNUFC JDP62 in this study were similar to those of the previously described T. beijingensis by Chen et al. [34]. However, the number of metulae are larger than those of T. beijingensis and smaller conidia [34]. In addition, differences in colony diameter were observed on MEA (35-39 mm) [34].

Discussion
Recent studies on the genera Aspergillus, Penicillium, and Talaromyces have been conducted in Korea, yet their distribution is not fully known, with various areas remain unexplored. In this study, a new species and eight new records are described and illustrated. The ITS is the DNA barcoding marker for the identification of fungi. However, many of the previous studies have shown that the only ITS is insufficient for Aspergillus, Penicillium, and Talaromyces identification as closely related species often share similar or identical sequences. The BenA, and CaM sequences have been used for the identification of these three genera, as this is the recommended identification marker [2,3,11]. In this study, the multigene phylogeny proposed by Houbraken et al. [11] allowed us to recognize a new species, and to identify eight recently described in the genera Aspergillus, Penicillium and Talaromyces. A new species described here belongs to section Citrina, series Westlingiorum. Penicillium aquadulcis is closely related to P. manginii, and are in one clade with Penicillium sp. SFC P0237 and Penicillium sp. SFC P0230. Penicillium sp. SFC P0237 and Penicillium sp. SFC P0230 were isolated from Rhododendron brachycarpum in Korea but there are no descriptions for these species [35]. Penicillium aquadulcis differs from P. manginii by having smaller colonies on CYA, MEA, and CREA. Conidial shape and production of sclerotia can be easily used to distinguish between Penicillium aquadulcis and P. manginii. Section Citrina currently includes 41 species and 9 series (ser. Gallaica, ser. Roseopurpurea, ser. Euglauca, ser. Citrina, ser. Paxillorum, ser. Sheariorum, ser. Sumatraensia, ser. Copticolarum and ser. Westlingiorum) [11]. Twentytwo species are currently accepted in series Westlingiorum and most of those species isolated from soil [36]. In this study, we isolated a novel species from freshwater.
Hawksworth [37] estimated that there are approximately 1.5 million fungal species on Earth. Around 3,000 species are known to be associated with aquatic habitats, with more than 600 species of ascomycetes reported from freshwater habitats [38,39]. The importance of these organisms in secondary metabolite production has also been recognized. However, the knowledge on freshwater fungi remains insufficient. Moreover, these species are less explored than terrestrial fungi.
In this study, both T. adpressus, and T. beijingensis were also found in freshwater. Talaromyces adpressus and T. beijingensis are known Talaromyces species, which were originally described from air in China. To the best of our knowledge, species from aquatic environments have been reported for the sections, Helici, Purpurei and Talaromyces [40,41]. Further studies are necessary to understand both the distribution and relevance of species in the section Talaromyces in aquatic environments.
Insects are known to host various fungi [16,42,43]. Recently, Barbosa et al. [42] reported an association of Penicillium and Talaromyces with stingless bees. Notably, we also isolated A. subversicolor from bee niche. Previous studies isolated A. subversicolor from coffee berries and as endophytes from macroalgae [30,44], suggesting that this species has a broad ecological niche. The occurrence and distribution of Aspergillus associated with insects in freshwater habitats remain unknown. Further studies on the relationship of fungi and insects are thus needed to understand the benefits that fungi may provide to their hosts. In addition, the genera Aspergillus, Penicillium, and Talaromyces are now known to produce secondary metabolites, warranting further studies on the biodiversity of these fungi and their potential applications in biotechnology.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This work was partly supported by the Project on Survey and Discovery of Indigenous Fungal Species of Korea funded by the NIBR and by the Project on Discovery of Fungi from Freshwater and Collection of Fungarium funded by the NNIBR of the Ministry of Environment (MOE).