New Species of Talaromyces (Fungi) Isolated from Soil in Southwestern China

Simple Summary Talaromyces species are distributed all around the world and occur in various environments, e.g., soil, air, living or rotten plants, and indoors. Some of them produce enzymes and pigments of industrial importance, while some cause Talaromycosis. Talaromyces marneffei, a well-known and important human pathogen, is endemic to Southeast Asia and causes high mortality, especially in HIV/AIDS patients and those with other immunodeficiencies. China covers 3 of the 35 global biodiversity hotspots. During the explorations of fungal diversity in soil samples collected at different sites of southwestern China, two new Talaromyces species, T. chongqingensis X.C. Wang and W.Y. Zhuang and T. wushanicus X.C. Wang and W.Y. Zhuang, were discovered based on phylogenetic analyses and morphological comparisons. They are described and illustrated in detail. Six phylogenetic trees of the sections Talaromyces and Trachyspermi were constructed based on three-gene datasets and revealed the phylogenetic positions of the new species. This work provided a better understanding of biodiversity and phylogeny of the genus. The results make the concepts of the two sections of Talaromyces well-established. The discovery will be beneficial for future evaluation of the potential usages and functions of the new species. Abstract Southwestern China belongs among the global biodiversity hotspots and the Daba Mountains are recognized as one of the priority conservation areas. During the exploration of fungal biodiversity from soil samples collected from Mount Daba, two species of Talaromyces were discovered as new to science based on phylogenetic analyses and morphological comparisons. Talaromyces chongqingensis sp. nov. is a sister taxon of T. minioluteus and T. minnesotensis in the section Trachyspermi; and T. wushanicus sp. nov., affiliated to the section Talaromyces, is closely related to T. cnidii and T. siamensis. The new species differ from their sisters in DNA sequences, growth rates, and morphological characteristics. Descriptions and illustrations of them are provided in detail.


Introduction
Talaromyces C.R. Benj. is a cosmopolitan genus occurring in various environments, e.g., soil, air, living or rotten plants, and indoors. Its beneficial and harmful effects on humans have been well documented. Enzymes and pigments produced by some species of the genus are of industrial importance, such as β-glucosidase produced by T. amestolkiae N. Yilmaz et al. [1] and T. cellulolyticus T. Fujii et al. [2], and red pigments by T. atroroseus N. Yilmaz et al. [3,4]. Talaromycosis caused by several species were also reported [5,6]. Among them, T. marneffei (Segretain et al.) Samson et al., endemic to Southeast Asia, is a well-known and important human pathogen causing high mortality in the absence of proper diagnosis and prompt treatment, especially in HIV/AIDS patients and those with other immunodeficiencies [7].
A total of 170 Talaromyces species were accepted and classified into seven sections according to a recent monographic study [8]. Moreover, T. albisclerotius B.D. Sun [9][10][11][12]. In the section (sect.) Trachyspermi Yaguchi and Udagawa, 30 species are commonly accepted; and in the sect. Talaromyces, the largest part of the genus, 75 species have been recognized.
During the explorations of fungal diversity in soil samples collected at different sites of Chongqing and Sichuan in southwestern China, two Talaromyces species belonging to the sections Talaromyces and Trachyspermi were further discovered as new to science based on phylogenetic analyses and morphological comparisons. They are described and illustrated in detail.

Fungal Materials
Cultures were isolated from soil samples collected from Chongqing and areas nearby in Sichuan Province in October 2020. Dried cultures were deposited in the Herbarium Mycologicum Academiae Sinicae (HMAS), and the living ex-type strains were preserved in the China General Microbiological Culture Collection Center (CGMCC).

DNA Extraction, PCR Amplification, and Sequencing
DNA was extracted from the cultures grown on PDA for 7 days using the Plant Genomic DNA Kit (DP305, TIANGEN Biotech, Beijing, China). Polymerase chain reaction (PCR) amplifications of the internal transcribed spacer (ITS), beta-tubulin (BenA), calmodulin (CaM) and RNA polymerase II second largest subunit (RPB2) gene regions were conducted with routine methods [14][15][16]. The products were purified and subject to sequencing on an ABI 3730 DNA Sequencer (Applied Biosystems, Bedford, MA, USA). Although the ITS region, the recommended standard DNA barcode for fungi, is not sufficient to discriminate the species of this genus, the sequences provided here will be helpful for other researchers in case of need.

Phylogenetic Analyses
The forward and reverse sequences newly generated in this study were assembled using Seqman v. 7.1.0 (DNASTAR Inc., Madison, WI, USA). The assembled sequences were deposited in GenBank. Previously described species from the corresponding sections, which were used for phylogenetic analyses, are listed in Tables 1 and 2. Newly generated sequences of this study are shown in Table 3. For each section, three datasets of BenA, CaM, and RPB2 were compiled. Sequences of each dataset (35 species for sect. Trachyspermi and 79 species for sect. Talaromyces) were aligned using MAFFT v. 7.221 [17], and then manually edited in BioEdit v. 7.1.10 [18] and MEGA v. 6.0.6 [19]. Maximum likelihood (ML) analyses were performed using RAxML-HPC2 [20] on XSEDE 8.2.12 on CIPRES Science Gateway v. 3.3 [21] with the default GTRCAT model. Bayesian Inference (BI) analyses were performed with MrBayes v. 3.2.5 [22]. Appropriate nucleotide substitution models and parameters were determined by Modeltest v. 3.7 [23]. The consensus trees were viewed in FigTree v. 1.3.1 (Available online: http://tree.bio.ed.ac.uk/software/figtree/ (accessed on 1 September 2015)). The type species of section Talaromyces served as outgroup taxon of the Trachyspermi tree and vice versa.

Phylogenetic Analysis
The characteristics of datasets used in the phylogenetic analyses are presented in Table 4. Phylogenetic analyses of the section Trachyspermi revealed that T. chongqingensis always grouped with T. minioluteus, T. minnesotensis, and T. udagawae, having strong statistic supports. In the BenA and CaM analyses (Figures 1 and 2), T. minioluteus was the closest sister of the new species; while T. minioluteus and T. minnesotensis were both closely related to T. chongqingensis in the RPB2 tree ( Figure 3). In the phylogenetic analysis of section Talaromyces based on the BenA dataset, T. wushanicus clustered with T. siamensis ( Figure 4); while T. cnidii and T. siamensis were closely related to the new species in the CaM and RPB2 analyses (Figures 5 and 6).

Taxonomy
Talaromyces chongqingensis X.C. Wang and W.Y. Zhuang, sp. nov., Figure 7. Colony characteristics: On CYA at 25 °C, after 7 days: colonies nearly circular, protuberant in centers; margins moderately wide, entire; mycelia white and yellow; texture velutinous; sporulation dense; conidia en masse yellowish green to dull green; soluble pigments light brown; exudates small, clear; reverse orange, buff at the margins but dark orange at centers. On YES at 25 • C, after 7 days: Colonies nearly circular, strongly protuberant in centers; margins moderately wide, entire; mycelia white and yellow; texture velutinous; sporulation moderately dense; conidia en masse pale green; soluble pigments light brown; exudates absent; reverse orange, yellow brown at the margins but dark orange at centers.
Notes: This species is phylogenetically close to T. minioluteus and T. minnesotensis, but differs from them in growth rate on CYA and MEA at 25 • C (Table 5) and pink hyphae present at the centers of colonies on MEA. The sequence data of the four cultures of this fungus are completely identical. Talaromyces wushanicus X.C. Wang and W.Y. Zhuang, sp. nov., Figure 8.  Colony characteristics: On CYA 25 • C, 7 days: Colonies nearly circular, protuberant in centers; margins narrow to moderately wide, nearly entire; mycelia white; texture velutinous; sporulation moderately dense; conidia en masse yellowish green; soluble pigments absent; exudates almost absent, sometimes very tiny, red, clear; reverse buff, orange to light brown at centers, but white and pink at periphery.
On CYA at 37 • C, after 7 days: Colonies irregular or nearly circular, protuberant in centers; margins moderately wide, nearly entire; mycelia white; texture velutinous; sporulation moderately dense; conidia en masse dull green to greyish green; soluble pigments absent; exudates absent; reverse buff.
On MEA at 25 • C, after 7 days: Colonies nearly circular, plain; margins wide, entire; mycelia yellow; texture velutinous; sporulation dense; conidia en masse yellowish green; soluble pigments absent; exudates almost absent, sometimes very tiny, hyaline, clear; reverse buff, but yellow to orange in centers.
On YES at 25 • C, after 7 days: Colonies nearly circular, deep, wrinkled, highly protuberant in centers; margins narrow to moderately wide, entire; mycelia white; texture velutinous; sporulation dense; conidia en masse yellowish green to dark green; soluble pigments absent; exudates absent, rarely red and clear; reverse white, yellow brown to light brown, rimose, or deeply concave in centers.
On PDA at 25 • C, after 7 days: Colonies nearly circular, plain, slightly protuberant in centers; margins moderately wide, entire; mycelia white; texture velutinous; sporulation dense; conidia en masse yellowish green; soluble pigments absent; exudates hyaline, clear, present at centers; reverse greyish white to greenish white, reddish brown at centers.
Notes: This species is closely related to T. cnidii and T. siamensis in the phylogenetic trees (Figures 4-6), but it differs from T. cnidii in obviously slower growth rate on CYA and YES at 25 • C and from T. siamensis by an obviously faster growth on MEA at 25 • C ( Table 5). Sequence comparisons indicate that the isolate CS17-04 has a one-base difference in ITS and a two-base difference in BenA from the other two strains. No morphological diversification was found among the strains.

Discussion
Of the 35 global biodiversity hotspots, 3 are located in southwestern China, consisting of Chongqing, Guizhou, Sichuan, Tibet, and Yunnan provinces [26]. Eight hotspot regions in the southwest of China were identified as priority conservation areas, including the Daba Mountains [27] where materials used for this study were gathered. Soil samples for floristic studies of fungi were collected from Chengkou, Wushan, and Wuxi counties in Chongqing and Wanyuan City in Sichuan. Although Talaromyces is a widespread genus and distributed in more than 27 provinces, cities, or regions of China [14], it has never been reported from the above areas.
In recent years, the number of new species of Talaromyces increased dramatically. There were 12 species recorded in Talaromyces sect. Trachyspermi and 36 ones in Talaromyces sect. Talaromyces in 2014 [24]. From 2018 to 2021, 13 additional species were discovered in the former section, and 20 new members were described in the latter. We are witnessing a trend: new fungal species are described at an accelerated rate.
Talaromyces species occur in diversified environments. When the information about the extype strains of more than 100 species in these two sections is gathered (Tables 1 and 2), it is found that soil is commonly the substrate. Fifty or so species were isolated from different kinds of soil, e.g., forest, cultivated, and swamp soil. Plant debris appears to be the second frequent source, which nearly 20 species inhabited. Four species were from humans and one, the well-known T. marneffei, from bamboo rat. Surprisingly, T. pinophilus was discovered on PVC, the third widely used plastic in the world, which is hard to biodegrade.
Among the 30 species accepted in Talaromyces sect. Trachyspermi, 6 were originally reported from China (Table 1). Moreover, 18 of the 75 species known in Talaromyces sect. Talaromyces were described based on the Chinese samples or specimens ( Table 2). These data surely demonstrate that China has a high fungal diversity. With more investigations conducted, we expect to discover more new species of this group of fungi.

Conclusions
The present work provides a better understanding of biodiversity and phylogeny of the genus. The results make the concepts of the two sections of Talaromyces well-established and more sophisticated. The discovery will be beneficial for future evaluation of the potential usages and functions of the new species.