New section and species in Talaromyces

Abstract Talaromyces is a monophyletic genus containing seven sections. The number of species in Talaromyces grows rapidly due to reliable and complete sequence data contributed from all over the world. In this study agricultural soil samples from Fujiang, Guangdong, Jiangxi, Shandong, Tibet and Zhejiang provinces of China were collected and analyzed for fungal diversity. Based on a polyphasic approach including phylogenetic analysis of partial ITS, BenA, CaM and RPB2 gene sequences, macro- and micro-morphological analyses, six of them could not be assigned to any described species, and one cannot be assigned to any known sections. Morphological characters as well as their phylogenetic relationship with other Talaromyces species are presented for these putative new species. Penicillium resedanum is combined in Talaromyces section Subinflati as T. resedanus.


Introduction
The genus Talaromyces used to accommodate sexual Penicillium species (Benjamin 1955). The generic concept has changed in the last decade due to changes in nomenclatural rules and results of phylogenetic studies. Before 2011, various studies showed that asexual reproducing Penicillium species classified in subgenus Biverticillium and the sexual Talaromyces species form a monophyletic clade distinct from Penicillium sensu stricto (LoBuglio et al. 1993;Berbee et al. 1995;Ogawa et al. 1997;Ogawa and Sugiyama 2000;Wang and Zhuang 2007;Houbraken and Samson 2011). In 2011, following the concepts of nomenclatural priority and single name nomenclature, Samson et al. (2011) transferred the majority of accepted Penicillium subgenus Biverticillium species to Talaromyces. A monograph of Talaromyces was provided based on a polyphasic species concept with seven sections Bacillispori, Helici, Islandici, Purpurei, Subinflati, Talaromyces and Trachyspermi (Yilmaz et al. 2014). This sectional classification was further supported by a four gene phylogeny ). The number of species in Talaromyces grows rapidly due to reliable and complete sequence data contributed from all over the world (Visagie et al. 2015;Crous et al. 2016Crous et al. , 2017Crous et al. , 2018Yilmaz et al. 2016a, b;Guevara-Suarez et al. 2017;Peterson and Jurjević 2017;Barbosa et al. 2018;Varriale et al. 2018;Rodríguez-Andrade et al. 2019;Rajeshkumar et al. 2019;Guevara-Suarez et al. 2020). It is noteworthy that in China many new species were discovered with another 19 new species reported Wang QM et al. 2016;Wang XC et al. 2016Jiang et al. 2018;Su and Niu 2018).
Talaromyces contains several species that are reported to cause infections in humans. Talaromyces marneffei has been exclusively associated with acquired immunodeficiency syndrome (AIDS) caused by human immunodeficiency virus (HIV) infections (Supparatpinyo et al. 1994;Limper et al. 2017). Other species like T. indigoticus, T. helicus, T. piceus, T. purpurogenus, T. radicus, T. rugulosus and T. verruculosus have been reported in superficial or disseminated, fatal infections (Horré et al. 2001;Santos et al. 2006;de Vos et al. 2009;Weisenborn et al. 2010;Tomlinson et al. 2011;de Hoog et al. 2014). Recently, four new members of Talaromyces were reported from clinical sources, and more studies are needed to complete the distribution and the relevance of these new fungi in human and animal disease .
In this study, we collected agricultural soil samples from Fujiang, Guangdong, Jiangxi, Shandong, Tibet and Zhejiang provinces in China. After isolation and identification, six Talaromyces species could not be assigned to any known species. A polyphasic approach including phylogenetic analysis of partial ITS, β-tubulin (BenA), calmodulin (CaM) and RNA polymerase II second largest subunit (RPB2) gene sequences and macro-and micro-morphological data were used to delimitate the new species and section in this genus.

Isolates
Soil samples were collected from six provinces from China as mentioned above. A general dilution-plate method was used to isolate fungi, bacteria and actinomycetes. As for fungi, Potato Dextrose Agar (PDA, Guangdong huankai microbiological technology co., LTD) and Rose Bengal Medium (RBM, Beijing luqiao technology co., Ltd) with antibiotics (tetracycline hydrochloride and chloramphenicol with the final concentration of 100 mg/ml) were used. Obtained strains were purified and sub-cultured on malt extract agar (MEA, Guangdong huankai microbiological technology co., Ltd). Reference strains used in this study were obtained from the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, the CBS culture collection and the working collection of the Applied and Industrial Mycology department (DTO), both housed at the Westerdijk Fungal Biodiversity Institute (Utrecht, the Netherlands). An overview of strains is listed in Table 1. For other strains used in the phylogenetic analyses, readers are referred to Chen et al. 2016;Crous et al. 2016Crous et al. , 2017Crous et al. , 2018Wang QM et al. 2016;Wang XC et al. 2016Yilmaz et al. 2016a, b;Guevara-Suarez et al. 2017;Peterson and Jurjević 2017;Barbosa et al. 2018;Su and Niu 2018;Varriale et al. 2018;Rajeshkumar et al. 2019;Rodríguez-Andrade et al. 2019;Guevara-Suarez et al. 2020.

DNA extraction, PCR amplification and sequencing
Strains were grown for 1 wk on MEA prior to DNA extraction. DNA was extracted using the Ultraclean TM Microbial DNA isolation Kit (MoBio, Solana Beach, U.S.A.) and stored at -20 °C. The ITS, BenA, CaM, and RPB2 genes were amplified and sequenced using methods and primers previously described Yilmaz et al. 2014).

Phylogenetic analysis
For sectional classification in Talaromyces, a four-gene phylogeny combining ITS, BenA, CaM and RPB2 sequences was used. Prior to combining the datasets, single gene alignments were generated using MAFFT v. 7 (Katoh et al. 2019), and then trimmed at both ends. Aligned datasets were subsequently concatenated using Mesquite v 3.6 (Maddison and Maddison 2018). For each section, single gene phylogenies were generated to determine the phylogenetic relationship among species. The most suitable substitution model was determined using FindModel (Posada and Crandall 1998). Bayesian analyses were performed with MrBayes v. 3.2 (Ronquist et al. 2012). The sample frequency was set to 100 and the first 25% of trees were removed as burn-in. Maximum likelihood analyses including 1000 bootstrap replicates were run using RAxML (Kozlov et al. 2019

Morphological analysis
Macroscopic characters were studied on Czapek yeast autolysate agar (CYA), CYA supplemented with 5% NaCl (CYAS), yeast extract sucrose agar (YES), creatine sucrose agar (CREA), dichloran 18% glycerol agar (DG18), oatmeal agar (OA) and malt extract agar (MEA; Oxoid malt) (Samson et al. 2010). Isolates were inoculated at three points on 90 mm Petri dishes and incubated for 7 d at 25 °C in darkness. Additional CYA plates were incubated at 30 and 37 °C, and an additional MEA plate was incubated at 30 °C. After 7 d of incubation, colony diameters were recorded. The colony texture, degree of sporulation, obverse and reverse colony colors, the production of soluble pigments and exudates were noted. Acid production on CREA is indicated by a change in the pH sensitive bromocresol purple dye, from a purple to yellow color in media surrounding colonies. For ascoma production, OA, MEA and CYA plates were incubated for up to four wks. Color codes used in description refer to Rayner (1970). Microscope preparations were made from 1 wk-old colonies grown on MEA. Production of ascomata, asci and ascospores was determined on 2-3 wk-old colonies on OA. Size of ascospores and conidia were measured without ornamentation. Lactic acid (60%) was used as mounting fluid and 96% ethanol was applied to remove the excess of conidia. A Zeiss Stereo Discovery V20 dissecting microscope and Zeiss AX10 Imager A2 light microscope equipped with Nikon DS-Ri2 cameras and software NIS-Elements D v4.50 were used to capture digital images.

Phylogeny
The individual ITS, BenA, CaM and RPB2 datasets consist of 653, 591, 782 and 802 characters, respectively, and were combined to study the relationship within Talaromyces. The most optimal model for each dataset is listed in Table 2. Eight well-supported lineages are present in the multigene phylogenic analysis (Fig. 1). Seven lineages agree with sectional classification by Yilmaz et al. 2014 and one lineage, represented by a new species described here (Talaromyces tenuis), could not be assigned to any known section. This lineage is sister to sections Talaromyces and Helici but cannot be assigned into any of them. Based on its phylogenetic and morphological peculiarity (see description below), the lineage is described as a new section named Tenues. Furthermore, five new species are distributed over three sections, T. brevis, T. rufus and T. aspriconidius in section Talaromyces; T. albisclerotius in section Trachyspermi and T. guizhouensis in section Subinflati.
In section Talaromyces, T. rufus and T. aspriconidius can be separated via each single gene phylogram. Talaromyces rufus is close to T. macrosporus based on BenA, CaM and RPB2 phylograms and forms a separate lineage in ITS phylogram. Talaromyces aspriconidius is close to T. primulinus based on RPB2 phylogram, but clusters with T. flavus based on BenA phylogram, and forms a separate lineage in CaM and ITS phylograms. Talaromyces brevis is closely related to T. liani, it can be differentiated via BenA, CaM and RPB2 phylograms, but not via ITS phylogram ( Fig. 2; Suppl. materials: 1-3).
In section Trachyspermi, T. albisclerotius can be well-separated in four single phylograms; this species clusters with T. diversus in BenA, CaM and RPB2 phylograms, and forms a separate lineage in ITS phylogram (Fig. 3;. Talaromyces guizhouensis is assigned in section Subinflati and P. resedanum also belongs to this section according to our multigene analysis. With the newly described T. tzapotlensis and T. omanensis the total number of taxa belonging to section Subinflati increased from two to five since it was established in 2014. Talaromyces omanensis shares same ITS, BenA and CaM sequences with T. resedanus CBS 184.90. Talaromyces guizhouensis is close to T. tzapotlensis and T. subinflatus in each single gene phylogram (Fig. 4, Suppl. materials: 7-9).    Phylogenetic analysis places Talaromyces section Tenues sister to sections Talaromyces and Helici (Fig. 1); however, statistical support for this relationship is lacking. Using a nine-gene sequence data set, Houbraken et al. (2020) confidently shows that Talaromyces sp. CBS 141840 (= T. tenuis, the sole representative of the section) is sister to sect. Purpurei and Trachyspermi. Section Trachyspermi species produce abundant red pigments (Yilmaz et al. 2014), while Talaromyces tenuis does not. Section Purpurei species generally grow rapidly on CYA and MEA, and usually produce synnemata after two to three weeks of incubation (Yilmaz et al. 2014).

Sect. Trachyspermi BenA
Etymology. Named after the type species of the section, Talaromyces tenuis. Diagnosis. Talaromyces tenuis produces hyaline, thin conidiophores, yellow mycelium on MEA and OA, and grows very restrictedly on CYA, YES and DG18.
Note. Talaromyces tenuis is phylogenetically distinct and is basal to species belonging to sections Talaromyces and Helici (Fig. 1). In a nine-gene phylogeny, it is sister to sections Purpurei and Trachyspermi (Houbraken et al. 2020). This species is characterized by hyaline, thin conidiophores, and grows very restrictedly on CYA, YES and DG18; colonies on MEA and OA have prominent yellow mycelia.
Notes. Talaromyces albisclerotius is characterized by the production of white sclerotia on OA after 1 wk incubation; these sclerotia remain sterile and no ascospores are observed after prolonged incubation up to eight wk. Talaromyces assiutensis and T. trachyspermus could produce white ascomata, but their ascomata mature after weeks and release ascospores (Yilmaz et al. 2014). Phylogenetically, T. albisclerotius clusters with T. diversus and T. brasiliensis, but T. diversus grows faster on MEA, and T. brasiliensis produces rough conidia (Yilmaz et al. 2014;Barbosa et al. 2018).
Etymology. Latin, brevis, refers to its short conidiophores. Diagnosis. Talaromyces guizhouensis grows poorly on CREA and DG18, does not produce synnemata as well as ascospores.
Notes. Section Subinflati previously contained two species namely T. subinflatus and T. palmae. These species do not resemble each other, although both grow poorly on CREA and DG18 (Yilmaz et al. 2014). Talaromyces tzapotlensis was included more recently (Peterson and Jurjević 2017) and we here expand this section with T. guizhouensis and T. resedanus. Like the other species in this section, T. guizhouensis also grows poorly on CREA and DG18. This species is phylogenetically related to T. subinflatus, but the latter grows very restrictedly on common media except MEA (Yilmaz et al. 2014). Talaromyces palmae produces indeterminate synnemata and short stipes (up to 85 μm) (Yilmaz et al. 2014) and these are not observed in T. guizhouensis. Furthermore, T. tzapotlensis grows faster on most media (e.g., 29-30 vs 8-9 mm on CYA; 10-11 vs 4-5 mm on DG18; 20-22 vs 2-3 mm on CREA, all diam. after 7 days  and T. resedanus does not grow on CREA and produces smaller conidia measuring 2-3 × 1.5-2 μm.
This species was introduced as Penicillium resedanum (McLennan et al., 1954). Yilmaz et al. (2014) listed it as doubtful species because the ex-type culture CBS 181.71 was not viable at that time. We requested the lyophilized culture of CBS 181.71 and CBS 184.90 deposited in nitrogen, and successfully resurrected them. The concatenated alignment and the single gene phylogenies proved its assignment in section Subinflati. Talaromyces omanensis described by Halo et al. (2019)   and thus displayed green colony. The monoverticillate conidiophores, size and shape of stipes, phialides and conidia of T. omanensis resemble those of T. resedanus, except that conidiophores of T. omanensis are rough under scanning electron microscope (SEM) (Halo et al. 2019). The photo plate of T. omanensis showed smooth conidiophores under microscope. Based on the molecular and morphological similarity, we considered T. omanensis a synonym of T. resedanus. Diagnosis. This species produces red, determinate synnemata and ellipsoidal, spiny ascospores measuring 5-6 × 4-5 μm.

Discussion
Previous studies showed that the genus Talaromyces comprises seven sections (Yilmaz et al. 2014). In this study, a comprehensive isolation of soil samples was carried out in China; one new section and six new species were described using a polyphasic approach. Talaromyces section Tenues is newly introduced and contains one species. In our phylogenetic analysis, this section is sister to sections Talaromyces and Helici, though statistical support is lacking. Houbraken et al. (2020) studied the relationships within the Eurotiales and confidently showed that the section is sister to Purpurei and Trachyspermi. Section Tenues is morphologically characterized by restricted growth on CYA, YES and DG18, slightly faster growth on MEA and OA and no growth on CREA. Based on these phenotypic characteristics, section Tenues species resemble section Trachyspermi species, but the species in section Trachyspermi are likely to produce abundant red pigments (Yilmaz et al. 2014), while Talaromyces tenuis doesn't. Talaromyces tenuis also produces thinner conidiophores, and it is interesting to find out whether this character is shared by other species that will be described in this section in the future. Three of our new species fall into section Talaromyces. This section was first introduced for species that produce yellow ascomata, which can occasionally be white, creamish, pinkish or reddish, and have yellow ascospores (Stolk and Samson 1972). Nowadays, this section is not limited to sexual species, but it still contains the largest number and highest ratio of sexual reproducing species in this genus. Among three new species, T. brevis and T. rufus produce yellow, spiny ascospores, the ascospores of T. brevis are smaller compared to its close relatives T. liani, and T. rufus can be easily distinguished by its red synnemata. Talaromyces aspriconidius is characterized by its strikingly roughened, globose conidia, but ascospores were not observed after long incubation.
Talaromyces albisclerotius is classified in section Trachyspermi. Species in section Trachyspermi show restricted growth on CYA, YES and DG18, grow slightly faster on MEA, and do not, or poorly grow, on CREA. Conidiophores are generally biverticillate and some species produce creamish white or yellow ascomata (Yilmaz et al. 2014). The morphology of T. albisclerotius matches these characters well; however, T. albisclerotius produces white sclerotia and does not produce ascomata and ascospores. Yilmaz et al. (2016c) speculated that Talaromyces species with no known sexual stage may actually be heterothallic. They successfully induced the sexual reproductive structures in T. amestolkiae, which was formerly described as an asexual taxon with black sclerotia. Further study is needed on T. albisclerotius to complete this hypothesis.
Talaromyces guizhouensis and T. resedanus belong to section Subinflati. This section previously contained two morphologically distinct species T. palmae and T. subinflatus. Talaromyces palmae produces short, biverticillate conidiophores and indeterminate synnemata, and T. subinflatus produces longer conidiophores, and grows more restrictedly on all tested media (Yilmaz et al. 2014). Talaromyces tzapotlensis was described by Peterson and Jurjević (2017), it grows well on CREA and DG18. Talaromyces omanensis was considered as a synonym of T. resedanus based on molecular and morphological similarity. The growth rate of T. guizhouensis falls somewhere in between; it is phylogenetically close to T. subinflatus and T. tzapotlensis. Talaromyces resedanus is the only monoverticillate species in this section.
Talaromyces species have a worldwide distribution and are isolated from a wide range of substrates. Soil is their main habitat, but new species were also isolated from indoor air, dust, clinical samples, plants, seed, leaf litter, honey, pollen and stingless bee nests (Sang et al. 2013;Visagie et al. 2014;Chen et al. 2016;Wang QM et al. 2016;Yilmaz et al. 2016a;Guevara-Suarez et al. 2017;Peterson and Jurjević 2017;Crous et al. 2018;Su and Niu 2018;Rodríguez-Andrade et al. 2019). These studies expanded our knowledge on the substrates where Talaromyces species can occur, but on the other hand demonstrated the complicated ecological function of this genus. In this study, one new section and six new species were identified from soil in China. Further research will focus on the Talaromyces diversity from a wide range of substrates.