New Species of Talaromyces (Trichocomaceae, Eurotiales) from Southwestern China

Species of Talaromyces are cosmopolitan and ubiquitous, and some are of industrial and medicinal importance. Species of Talaromyces have been successively reported in China. During our examinations of samples collected from southwestern China, two new species belonging to Talaromyces sect. Talaromyces were further discovered based on phylogenetic analyses and morphological comparisons. Talaromyces ginkgonis sp. nov., isolated from a partially colonized fruit of Ginkgo biloba, differs from closely-related fungi in the combination of conidia ellipsoidal, smooth and 3.5−4 × 2−3 μm, no growth on CYA at 37 °C and sequence divergences; T. shilinensis sp. nov. is distinguished from its related allies in the combination of smooth conidia, colonies 10−11 mm diameter on CYA at 25 °C and sequence differences. Detailed descriptions and illustrations of the new taxa are given.


Introduction
Species of Talaromyces C.R. Benj. are cosmopolitan and ubiquitous, inhabiting soil, air, indoor environments, rotten food, plant debris, healthy plant as endophytes, insects, and immunodeficient humans. The beneficial and the harmful effects of Talaromyces have been well documented [1].
Seven sections have been established and widely accepted in the genus Talaromyces: Bacillispori, Helici, Islandici, Purpurei, Subinflati, Talaromyces, and Trachyspermi [2,3]. A novel section was recently proposed as sect. Tenues [4]. A total of 171 species were compiled in the genus and listed in the latest monograph [3]. Furthermore, 26 new taxa were afterwards noted [1,[4][5][6][7][8][9][10][11][12]. Twenty of them are from Asia: T. albisclerotius B.D. Sun  Southwestern China shows various climates, altitudes, and vegetations, and it is rich in fungal biodiversity. Two species from soil in Chongqing were just described [1]. Along with more samples isolated from the area being examined, two additional new species belonging to Talaromyces sect. Talaromyces were further discovered based on phylogenetic analyses and morphological comparisons. Detailed descriptions and illustrations of the new taxa are provided.

Fungal Materials
The new species were associated with fungal (Pseudocosmospora sp.) or plant (Ginkgo biloba L.) materials collected in southwestern China (Sichuan and Yunnan provinces) during 2016-2017. Dried cultures were deposited in the Herbarium Mycologicum Academiae Sinicae (HMAS, Beijing, China), and the living ex-type strains were preserved in the China General Microbiological Culture Collection Center (CGMCC, Beijing, China).

Molecular Experiments
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 [1,[14][15][16]. The products were purified and subjected to sequencing on an ABI 3730 DNA Sequencer (Applied Biosystems, Bedford, MA, USA). Although the ITS region is proposed as the universal DNA barcode for fungi, it is not sufficient to distinguish species of Talaromyces. The ITS sequences provided in this study might be helpful for other researchers in case of need.

Phylogenetic Analyses
The forward and the 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. Sequences used for phylogenetic analyses were listed in Table 1. Sequences of each of the three separate datasets (BenA, CaM, and RPB2) and those that were combined 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 determined using RAxML-HPC2 [20]

Phylogenetic Analysis
To infer the phylogeny of Talaromyces sect. Talaromyces and to determine the positions of the new species, three separate datasets (BenA, CaM and RPB2) and those that were combined were compiled and analyzed. Detailed characteristics of the datasets are listed in Table 2. In the BenA phylogeny ( Figure S1), the strain 10725 was clustered with T. aspriconidius, T. calidicanius, T. duclauxii, T. flavus, T. haitouensis, and T. marneffei; and XCW_SN259 was grouped with T. kabodanensis and T. primulinus. In the CaM tree ( Figure S2), 10725 showed as a distinct lineage, while XCW_SN259 was a sister taxon of T. primulinus. In the RPB2 phylogeny ( Figure S3), the position of 10725 was similar to that shown in the BenA phylogeny with relatively weak supports, while the sister relationship between T. primulinus and XCW_SN259 was confirmed as that in the CaM phylogeny. In the phylogenetic tree of the combined three-gene dataset (Figure 1), the position of 10725 was identical with the BenA and RPB2 trees and that of XCW_SN259 was consistent in of all the trees (Figures 1  and S1-S3).
On PDA 25 • C, 7 days: Colonies nearly circular to irregular, plain; margins wide, irregular; mycelia white; texture velutinous; sporulation dense; conidia en masse yellowish green to vivid green; soluble pigments absent; exudates absent; reverse usually pink at centers, green to buff at periphery, and white at margins.

Discussion
Forty-three species of the Talaromyces have been reported as new to science based on materials collected from China. They are distributed all over the country, especially in southwestern regions, for example, T. chongqingensis and T. wushanicus are from Chongqing, T. albisclerotius, T. guizhouensis, T. penicillioides, T. resinae, T. rosarhiza, and T. tenuis are from Guizhou, T. ginkgonis is from Sichuan, T. neofusisporus and T. qii are from Tibet, and T. aspriconidius, T. aureolinus, T. bannicus, T. rufus, T. shiliensis, and T. yunnanensis are from Yunnan [1,4,6,[9][10][11]. This proves that southwestern China is one of the global biodiversity hotspots. In northern China, 13 species were recorded from Beijing; in eastern parts of the country, 9 were from Jiangsu, Shandong, Shanghai, Taiwan, and Zhejiang; and a few species were occasionally found in the south, central, and northeast. This might be due to the frequency of investigations, climates, richness of plants, as well as human activities. We certainly expect to discover more species of the group in unexplored regions and even in surveyed areas in different seasons.
Along with the joining of the two new species, Talaromyces sect. Talaromyces currently possesses up to 86 species around the world. Forty species were originally described as being from Asia, of which 29 are from China, four are from Japan, two are from South Korea and Thailand, respectively, and only one was reported in India, Iran, and Vietnam; 18 taxa are from North America, including 14 from the USA and a single one from Canada, Cuba, Mexico, and Panama; 12 species are distributed in Europe (France, Italy, Netherlands, Spain, UK); six are reported in South America (Brazil, Colombia, and Ecuador); five are from Africa (Ghana and South Africa); and four are from Oceania (Australia and New Zealand). Concerning the known distribution of the genus, one may easily imagine that the biodiversity of Talaromyces may have been underrated, although it is well recognized in areas of East Asia and North America, intensive excursions covering a broad range of areas in the world should be suggested to have a better understanding of the biodiversity of this group.