Four novel Talaromyces species isolated from leaf litter from Colombian Amazon rain forests

Various Talaromyces strains were isolated during a survey of fungi involved in leaf litter decomposition in tropical lowland forests in the Caquetá and Amacayacu areas of the Colombian Amazon. Four new Talaromyces species are described using a polyphasic approach, which includes phenotypic characters, extrolite profiles and phylogenetic analysis of the internal transcribed spacer region (ITS) barcode, and beta-tubulin (BenA) and calmodulin (CaM) gene regions. Talaromyces amazonensis sp. nov., T. francoae sp. nov. and T. purgamentorum sp. nov. belong to Talaromyces section Talaromyces, and T. columbiensis sp. nov. is located in section Bacillispori. The new species produce several bioactive compounds: T. amazonensis produces the potential anticancer agents duclauxin, berkelic acid and vermicillin, and T. columbiensis produces the effective anticancer agent wortmannin (together with duclauxin). In addition to the new species, T. aculeatus and T. macrosporus were isolated during this study on leaf litter decomposition.


Introduction
Fungi play an important role in forest ecosystems, as they are involved in processes such as decomposition of leaf litter. Leaf decomposition contributes to nutrient cycling, in which a large proportion of nutrients from net primary production is returned to the forest floor (Maggs 1985). A number of papers have recently been published on fungal biodiversity in the Colombian Amazon region, reporting new species and new registers. Studies in this region revealed 248 species of macrofungi, but a large number of collections have remained unidentified (López-Quintero et al. 2012;Vasco-Palacios and Franco-Molano 2013). Several new taxa of micro-and macrofungi have been published-for instance, new species of Penicillium and Trichoderma López-Quintero et al. 2013;Vasco-Palacios and Franco-Molano 2013;Vasco-Palacios et al. 2014).
Talaromyces species have a worldwide distribution and are isolated from a wide range of substrates. The genus was initially described for teleomorphic species with a Penicillium or Penicillium-like anamorph that produces soft-walled ascomata covered by interwoven hyphae. Based on phylogenetic, phenotypic and extrolite data, and following the concept of single-name nomenclature, Samson et al. (2011) transferred all accepted species of Penicillium subg. Biverticillium to Talaromyces. The Talaromyces monograph of Yilmaz et al. (2014) covered 88 species, and the genus currently includes 103 species (Visagie et al. 2015;Romero et al. 2016;Wang et al. 2016;Yilmaz et al. 2016).
Numerous microfungi were isolated from decomposing leaf litter as part of a project to investigate their diversity and their role in the regeneration process of tropical lowland forests. Barcoding using ITS sequences (Schoch et al. 2012) revealed the presence of several Talaromyces species, and their taxonomic status is the subject of this report. Four new species are proposed and described using a polyphasic taxonomy, which includes phenotypic characters, analysis of extrolites and multiple gene phylogenies of internal transcribed spacer region (ITS), beta-tubulin (BenA) and calmodulin (CaM) gene regions.

Strains
The strains were isolated from leaf litter as described in Houbraken et al. (2011). In short, small particles from fresh or 4-6 month-old leaf litter were washed and placed on 2 % water agar. The strains were obtained from three forests in Colombia Amazonia, namely mature forests in Araracuara (Middle Caquetá) and Amacayacu (Amazonas), and a Pseudomonotes tropenbosii (Dipterocarpaceae) forest in Peña Roja (Middle Caquetá). The litterbag studies were carried out during February 2000 and July 2001 for the plots located in the Caquetá region and between August 2003 andSeptember 2004 in the plots located in the Amazonas. The newly obtained isolates and strains used for comparison are listed in Table 1, together with their origin, substrate information and GenBank accession numbers for ITS, BenA and CaM genes. Strains of the putative new Talaromyces species used in this study were deposited in the culture collection of CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands; the Center for Microbial Biotechnology at the Department of Systems Biology, Technical University of Denmark, Lyngby, Denmark (IBT); and the working collection of the Department of Applied and Industrial Mycology (DTO), housed at CBS-KNAW. The type material of the new taxa is preserved at the herbarium of the University of Antioquia (HUA).

DNA extraction, PCR amplification and sequencing
DNA extractions were made from strains grown for 7-14 days on MEA using the UltraClean ® Microbial DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA), and extracted DNA was stored at −20°C. ITS, CaM and BenA gene regions were amplified and sequenced using previously described methods Yilmaz et al. 2014).

Phylogeny
Sequence contigs were assembled in SeqMan v. 9.0.4 (DNASTAR, Inc., Madison, WI, USA). The newly generated sequences were included in a data set including sequences obtained from Yilmaz et al. (2014) and supplemented with sequences of new species that were subsequently described (Visagie et al. 2015;Wang et al. 2016;Yilmaz et al. 2016). The dataset for each gene was aligned using the MUSCLE software included in the MEGA v. 6.0.6 software package (Tamura et al. 2013). The aligned ITS, BenA and CaM data were analyzed using the maximum likelihood (ML) method. The model for ML was selected based on the Akaike information criterion (AIC) calculated in MEGA 6.06. The analysis was performed by calculating an initial tree using BIONJ, and the subsequent heuristic using Nearest-Neighbour-Interchange (NNI). Bootstrap support was calculated using 1000 replicates.

Morphological analysis
Strains were morphologically studied on different media under different growth conditions. Cultures were inoculated onto Czapek yeast extract 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) at three points on 90-mm Petri dishes and incubated for 7 days at 25°C in darkness. All media were prepared as described by Visagie et al. (2014). Additional MEA and CYA plates were incubated at 30°C and 37°C for 7 days in darkness. After incubation, the diameters of the colonies on the various media were measured. The density of sporulation, obverse and reverse colony colours, and the production of soluble pigments were noted. Colony colour codes refer to Kornerup and Wanscher (1967). Colonies were photographed with a Canon EOS 400D camera. Species were characterized microscopically by preparing slides from MEA. Lactic acid was used as mounting fluid, and excess amounts of conidia were washed with ethanol. Specimens were examined using a ZEISS AxioSkop 2 plus microscope, and the Nikon NIS-Elements D software package was used for capturing photographs and taking measurements.

Extrolites
The strains isolated during this study were analyzed by highperformance liquid chromatography (HPLC) with diode array detection as described by Houbraken et al. (2012). Three agar plugs from the Talaromyces isolates grown on CYA, MEA, YES and OA at 25°C for 7 days were extracted as described by Smedsgaard (1997). The detected metabolites were identified by comparison with standards according to Kildgaard et al. (2014) and Klitgaard et al. (2014).     Colony texture and ascomata on OA after 2-week incubation. c-e Conidiophores. f Asci and ascospores. g Ascospores. h Conidia. Scale bar: h = 10 μm applies to c-h

Results
Diversity of Talaromyces species ITS barcoding of the isolated fungi revealed the presence of various Talaromyces species among the other fungi isolated from the litterbags. Two known species, T. aculeatus and T. macrosporus, were isolated from the samples, and several other isolates seemed to represent species unknown to science. They are further studied in detail below and described as new species Talaromyces amazonensis, T. columbiensis, T. francoae and T. purgamentorum.

Phylogeny
The phylogenies presented in Fig. 1 show that the isolates studied here are accommodated in Talaromyces section Talaromyces (i.e., T. amazonensis, T. francoae and T. p u rg a m e n t o r u m ) and section B a c i l l i s p o r i (T. columbiensis). The concatenated data set was 1348 bp in length (ITS 458 bp; BenA and CaM 445 bp). The Tamura 3-parameter (T92) with gamma distribution (+G) and invariant sites (+I) was found to be the most suitable model for the ML analysis of the combined tree (Fig. 1). For the single gene trees, the T92+G+I model was found to be the most suitable for the ML analysis of the ITS alignment, while the Kimura 2-parameter (K2)+G+I was the most suitable model for both BenA and CaM data sets. ML trees were used to present the phylogenetic data, with bootstrap values (bs) shown near the nodes of the phylogram (Fig. 1). The BenA and CaM data sets had sufficient discriminatory power to differentiate the new species from other recognized species. The ITS barcode of T. francoae (CBS 113134 T ) was identical to that of T. apiculatus (CBS 312.59 T ).
Talaromyces columbiensis (CBS 113151 T ) is located close to T. unicus, and T. francoae (CBS 113134 T ) is closely related to T. kendrickii in all phylogenies. Both T. amazonensis (CBS 140373 T ) and T. purgamentorum (CBS 113145 T ) shift their positions between phylogenies, making it difficult to determine their closest relatives.

Morphology
The new taxa share similarities with other known species, but can also be differentiated by various characters. Detailed descriptions of the new species are given in the Taxonomy section, and the differences from the other Talaromyces species are summarized in the Discussion. In short, T. amazonensis (CBS 140373 T ) produced yellow ascomata on OA at 25 and 30°C after 1-2 weeks of incubation (Fig. 2), and its ascospores resembled those of T. flavus, T. muroii, T. liani and T. thailandensis. Talaromyces columbiensis (CBS 113151 T ) produced yellow ascomata on MEA, smooth ascospores with a single equatorial ridge, and has no growth at 37°C (Fig. 3). No sexual state was observed in T. francoae (CBS 113134 T ) or T. purgamentorum (CBS 113145 T ). Both species produced ampulliform phialides with a tapering neck and spiny, globose conidia (Figs. 4 and 5). They were not able to grow at 37°C and lacked acid production on CREA. Talaromyces purgamentorum exhibited restricted growth on DG18 and YES compared to T. francoae.

Extrolites
The species described here produce various extrolites, all of which are previously found in Talaromyces (Frisvad et al. 1990). Especially common are mitorubrinic acid, alternariol and the penicillides (Table 2). Details on the extrolites found in each species are given in the species descriptions.

Discussion
This study focused on four novel Talaromyces species isolated from leaf litter in several Colombian lowland rain forests. The multiple gene phylogenies (ITS, BenA and CaM) resolved strains into four distinct lineages that represent species. Based on a multigene phylogeny (ITS, BenA and RPB2), Yilmaz et al. (2014) divided the genus into seven clades and provided a sectional classification. Three of the species from this study are located in Talaromyces section Talaromyces, whereas one species (T. columbiensis) falls in section Bacillispori. Previous studies showed that ITS, which is the accepted DNA barcode for fungi (Schoch et al. 2012), is not sufficient to distinguish all Talaromyces species (Yilmaz et al. 2012;Frisvad et al. 2013;Manoch et al. 2013;Yilmaz et al. 2014). As a result, Yilmaz et al. (2014) proposed the use of BenA as a suitable secondary barcode marker for identification of Talaromyces isolates. With the exception of T. francoae, however, all newly described species could be identified by using ITS alone.
BenA and CaM data sets showed that all novel species are clearly distinct from previously described Talaromyces species. Visagie et al. (2015) revised Talaromyces species producing ampulliform phialides and rough-walled, globose conidia, and recognized eight species. Except for T. kendrickii, all species could grow at 37°C. In this study, T. francoae and T. purgamentorum have ampulliform phialides and rough-walled, globose conidia. Neither species grows at 37°C, and neither produces acid on CREA. These two characters of the new species are shared with T. kendrickii. However, on CYA at 25°C, T. kendrickii (25-31 mm) grows faster than T. francoae (8-10 mm) and T. purgamentorum (19-20 mm). Talaromyces francoae differs from T. purgamentorum by their growth rates on CYA and YES. The former species exhibits restricted growth on CYA (8-10 mm) and moderately fast growth on YES (20-22 mm), while T. purgamentorum attains a diameter on CYA of 19-20 mm and 10-12 mm on YES. Talaromyces amazonensis produces yellow ascomata with spiny, ellipsoidal ascospores, and these characters are common in the genus Talaromyces. Ascospores of T. amazonensis resemble those of T. flavus, T. muroii, T. thailandensis, and T. liani. Both T. flavus and T. muroii grow more restrictedly on CYA than T. amazonensis (9-16 mm vs. 30-38 mm). Talaromyces amazonensis differs from T. thailandensis by its ability to grow at 37°C. The main difference between T. liani and T. amazonensis is the ascoma colour. Talaromyces liani has orange-red ascomata, whereas the ascomata of T. amazonensis are yellow. Talaromyces columbiensis produces abundant yellow ascomata and smooth-walled ascospores with a single ridge on MEA after 2 weeks of incubation at 25°C. This species is phylogenetically closely related to T. unicus, which also produces ascospores with a single equatorial ridge. However, T. unicus differs by the production of ascospores with a very rough to spiny wall. Talaromyces stipitatus also produces ellipsoidal, smooth-walled ascospores with a single equatorial ridge. However, this species grows faster on standard agar media (CYA, MEA, OA and YES) and is able to grow on CYA incubated at 37°C.
Talaromyces is an important genus in biotechnology and in medical and food mycology (Yilmaz et al. 2014). Talaromyces species are isolated worldwide from many kinds of substrates, including house dust, decaying leaves, soil, air, compost, food products, humans and animals. The fungi are considered key players in leaf litter decomposition because of their ability to produce a wide range of extracellular enzymes (de Boer et al. 2005), and some Talaromyces species also produce cellulosedegrading enzymes (Pol et al. 2012;Maeda et al. 2013;Fuji et al. 2014). In this study, we isolated six Talaromyces species from leaf litter in the Colombian Amazon region. Two of them, T. aculeatus and T. macrosporus, have been previously described. The other four species are new to the literature, and thus far found only in decomposed leaf litter in Amazonia, Colombia.
The new Talaromyces species were efficient producers of a series of anticancer compounds. Talaromyces amazonensis in particular produced three different anticancer compounds: duclauxin, berkelic acid and vermicillin. It would be interesting to determine whether these anticancer compounds have a synergistic effect when combined. Another interesting compound found in T. columbiensis was wortmannin, also an anticancer compound, with several additional biological activities.
Etymology: From Amazonas, the department from which the species was isolated.
Diagnosis: Moderately fast growth on CYA, MEA, YES and OA at 25°C, restricted growth on DG18 at 25°C, moderate acid production on CREA. Abundant yellow ascomata produced on MEA and OA after 1-2 weeks at 25°C with broadly ellipsoidal, thick-walled, spiny ascospores.