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 (Houbraken et al. 2011; 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.

Material and methods

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 and September 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).

Table 1 Talaromyces strains used in the phylogenetic analysis
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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 (Visagie et al. 2014; 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. Figure plates were prepared in Adobe® Photoshop® CS6.

Extrolites

The strains isolated during this study were analyzed by high-performance 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).

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. purgamentorum) and section Bacillispori (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 113134T) was identical to that of T. apiculatus (CBS 312.59T).

Fig. 1
figure 1figure 1

Phylogenies based on the ITS, BenA, CaM and combined data set showing the relationship between the new species described in the study and other members of Talaromyces sections Talaromyces and Bacillispori. Strains of known species (T. aculeatus and T. macrosporus) isolated in the course of this study are marked with asterisks in ITS and BenA phylograms. Strains belonging to new species are indicated by bold text. Support in nodes are indicated by thickened branches for bootstrap values ≥ 70, % and T indicates ex-type strains

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Talaromyces columbiensis (CBS 113151T) is located close to T. unicus, and T. francoae (CBS 113134T) is closely related to T. kendrickii in all phylogenies. Both T. amazonensis (CBS 140373T) and T. purgamentorum (CBS 113145T) 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 140373T) 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 113151T) 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 113134T) or T. purgamentorum (CBS 113145T). 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.

Fig. 2
figure 2

Morphological characters of Talaromyces amazonensis (CBS 140373T). a Colonies from left to right (top row) CYA, MEA, DG18 and OA; (bottom row) CYA reverse, MEA reverse, YES and CREA. b 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

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Fig. 3
figure 3

Morphological characters of Talaromyces columbiensis, CBS 113151T. a Colonies from left to right (top row) CYA, MEA, DG18 and OA; (bottom row) CYA reverse, MEA reverse, YES and CREA. b–d Conidiophores. e, f Asci and ascospores. g Conidia. Scale bar: f = 10 μm applies to b–g

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Fig. 4
figure 4

Morphological characters of Talaromyces francoae, CBS 113134T. a Colonies from left to right (top row) CYA, MEA, DG18 and OA; (bottom row) CYA reverse, MEA reverse, YES and CREA. b–f Conidiophores. g Conidia. Scale bar: g = 10 μm applies to b–g

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Fig. 5
figure 5

Morphological characters of Talaromyces purgamentorum, CBS 113145T. a Colonies from left to right (top row) CYA, MEA, DG18 and OA; (bottom row) CYA reverse, MEA reverse, YES and CREA. b–f Conidiophores. g Conidia. Scale bar: g = 10 μm applies to b–g

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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.

Table 2 Production of extrolites by the Talaromyces species isolated in this study
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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 cellulose-degrading 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.

Taxonomy

Talaromyces amazonensis Yilmaz, López-Quintero, Vasco-Pal., Frisvad & Houbraken, sp. nov. Mycobank MB816230. Fig. 2.

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.

Typus: Colombia, dept. Amazonas, Araracuara community in the Middle Caquetá, ex mixed leaves in litterbag, after 6 months of decomposition in a mature forest, isolated in July 2000, C. López-Quintero (holotype HUA 197223; culture ex-type CBS 140373 = IBT 23215 = DTO 093-F9).

ITS barcode: KX011509 (alternative markers: BenA = KX011490; CaM = KX011502).

Colony diameter 7 days (mm): CYA 30–38; CYA 30 °C 35–42; CYA 37 °C 19–22; MEA 40–45; MEA 30 °C 50–55; DG18 20–25; CYAS No growth; OA 30–45; CREA 12–18; YES 35–42.

Colony characters: CYA 25 °C, 7 days: Colonies slightly raised at centre, slightly sulcate; margins low, plane, entire (2 mm); mycelium white and in the centre brownish red due to the pigmentation in reverse; texture floccose; sporulation absent; soluble pigments absent; exudates absent; reverse light yellow (4A4) with dark red (10C8) dots. MEA 25 °C, 7 days: Colonies slightly raised at centre, plane; margins low, plane, entire (2 mm); mycelium white; texture floccose; sporulation sparse to moderately dense at centre, conidia en masse dull green (27D3); soluble pigments absent; exudates absent; reverse greyish orange (5B6–6B6). YES 25 °C, 7 days: Colonies sunken at centre, slightly sulcate; margins low, plane, entire (2–3 mm); mycelium white, in some parts pastel-red and in the centre yellowish brown (5E4) due to the reverse pigment; texture floccose; sporulation absent; soluble pigments absent; exudates absent; reverse light yellow (4A4) with dark red (10C8) dots. DG18 25 °C, 7 days: Colonies low, plane; margins low, plane, entire (1–2 mm); mycelium white; texture velvety; sporulation dense, conidia en masse dull green (25D5–26D4); soluble pigments absent; exudates absent; reverse greenish white to greenish grey (28A2–28B2). OA 25 °C, 7 days: Colonies low, plane, yellow ascomata also at 30 °C abundant; margins low, plane, entire (4–5 mm); mycelium yellow; texture floccose; sporulation absent; soluble pigments absent; exudates absent; reverse pastel-yellow. CREA 25 °C, 7 days: weak growth, moderate acid production.

Micromorphology: Conidiophores biverticillate, sometimes monoverticillate; stipes smooth-walled, 30–220 × 2–3 μm; metulae divergent, two to five, 7.5–12 × 2.4–3.8 μm; phialides acerose, two to five per metula, 8–13 × 2–3 μm; conidia smooth-walled, ellipsoidal, 2.5–4(–6) × 2–3 μm. Ascomata bright yellow, globose to subglobose, abundantly ripening within 1–2 weeks on OA at 25 °C, covered with a few layers of well-developed networks of yellow hyphae; asci broadly globose to subglobose, 9–13 × 8–12 μm; ascospores broadly ellipsoidal, thick-walled, spiny, 3–5.5 × 2–4 μm.

Extrolites: Talaromyces amazonensis produces berkelic acid, duclauxin, mitorubrinic acid and vermicillin. Berkelic acid was first found as an anticancer agent in T. ruber (Stierle et al. 2006). The anticancer agent duclauxin was first found in T. duclauxii, but this compound has also been reported from T. macrosporus (Shibata et al. 1965; Frisvad et al. 1990; Bryant et al. 1993). Duclauxin was also reported from Penicillium herquei and P. manginii (Bryant et al. 1993; Cao et al. 2015), but the strains used had been misidentified, and the producers were Talaromyces species as well. Vermicillin is the third anticancer agent from T. amazonensis (Fuska et al. 1979). Mitorubrinic acid is a common azaphilone metabolite in the genus Talaromyces (Frisvad et al. 1990).

Talaromyces columbiensis Yilmaz, López-Quintero, Vasco-Pal., Frisvad & Houbraken, sp. nov. Mycobank MB 816231. Fig. 3.

Etymology: From Colombia, the country where the species was isolated.

Diagnosis: Restricted growth on CYA, MEA, YES, OA and DG18 at 25 °C and no growth at 37 °C. Yellow ascomata abundant on MEA after 2 weeks at 25 °C with ellipsoidal, smooth, single equatorial ridge ascospores.

Typus: Colombia, Amazonia, Natural Park Amacayacu, ex litterbag containing fresh mixed leaves from mature forest, isolated in September 2004, C. López-Quintero (holotype HUA 197225; cultures ex-type CBS 113151 = IBT 23206 = DTO 058-F3).

ITS barcode: KX011503 (alternative markers: BenA = KX011488; CaM = KX011499).

Colony diameter 7 days (mm): CYA 15–17; CYA 30 °C 18; CYA 37 °C No growth; MEA 15–17; MEA 30 °C 16–17; DG18 5; CYAS 3; OA 15–17; CREA 8–10; YES 14–15.

Colony characters: CYA 25 °C, 7 days: Colonies low, sulcate; margins low, plane, entire (1 mm); mycelium white and yellow; texture floccose; sporulation sparse; soluble pigments absent; exudates absent; reverse yellowish brown (5F5) centre fading to brownish orange to yellowish brown (5C5–5D5–5E5). MEA 25 °C, 7 days: Colonies raised at centre, sulcate; margins low, plane, entire (1 mm); mycelium dominantly yellow, in the margins white; texture floccose; sporulation sparse, conidia en masse greyish green (28C5–28D5); soluble pigments pale red; exudates absent; reverse brown (7E6) fading to reddish orange (7B7). YES 25 °C, 7 days: Colonies raised at centre, sulcate; margins low, plane, entire (1 mm); mycelium white, brownish orange to light brown (7C5–7D5); texture floccose; sporulation absent to sparse; soluble pigments absent; exudates absent; reverse brownish orange (5C5) centre fading to greyish orange (5B5). DG18 25 °C, 7 days: Colonies covered with sterile white mycelium; reverse yellowish white (4A2). OA 25 °C, 7 days: Colonies raised at centre, slightly sulcate; margins low, plane, entire (1–2 mm), no transparent zone; mycelium white and yellow; texture loosely funiculose to velvety; sporulation moderately dense at centre, conidia en masse dull green (26D4); soluble pigments absent; exudates small, clear droplets; reverse pastel orange-yellow. CREA 25 °C, 7 days: Weak growth, weak acid production.

Micromorphology: Conidiophores biverticillate; stipes smooth-walled, (40–) 50–130 (–145) × 2.4–3 μm; metulae divergent, three to six, 8.5–11.5 × 2.4–3.5 μm; phialides acerose, sometimes atypical, two to six per metula, 7–9.5 × 1.5–2.5 μm; conidia smooth-walled, ovoid to ellipsoidal, 2.5–3 × (1.8–) 2–2.5 μm. Ascomata bright yellow, globose to subglobose, ripening within 1–2 weeks on OA at 25 °C, covered with layers of well-developed networks of yellow hyphae; asci broadly globose to subglobose, 7–9 × 7.5–10 μm; ascospores ellipsoidal, finely rough to spiny, usually with a single equatorial ridge, 2.5–4.2 (–5) × 2.5–3.5 μm.

Extrolites: Talaromyces columbiensis produced duclauxin and several related naphthalenones, but also mitorubrins and wortmannin. Wortmannin was originally discovered as an antifungal agent, but it is also a potent enzyme inhibitor of regulatory kinases and is thus an interesting anticancer drug candidate (Wipf and Halter 2005). Wortmannin was originally reported from T. wortmannii and T. flavus (Brian et al. 1957), but these two species are not able to produce wortmannin (Yilmaz et al. 2014). It is thus interesting that T. columbiensis can produce this compound. Wortmannin has also been mentioned as a hemorrhagic mycotoxin (Abbas and Mirocha 1998) and an inhibitor of aflatoxin biosynthesis (Lee et al. 2007).

Talaromyces francoae Yilmaz, López-Q., Vasco-Pal. & Houbraken, sp. nov. Mycobank MB 816232. Fig. 4.

Etymology: named after Franco, the family name of Dr. Ana Esperanza Franco-Molano, who made major contributions to Colombian mycology.

Diagnosis: Conidiophores biverticillate with ampulliform phialides that taper to a very thin neck producing rough-walled, verrucose conidia. Lack of acid production on CREA and absence of growth on CYA at 37 °C. Restricted growth on CYA at 25 °C (8–10 mm).

Typus: Colombia, Dept. Amazonas, Peña Roja, ex leaf litter from 4-month old litterbag in Pseudomonotes tropenbosii (Dipterocarpaceae) forest, isolated in March 2000, C. López-Quintero (holotype HUA 197224; culture ex-type CBS 113134 = IBT 23221 = DTO 056-D9).

ITS barcode: KX011510 (alternative markers: BenA = KX011489; CaM = KX011501).

Colony diameter 7 days (mm): CYA 8–10; CYA 30 °C 29–30; CYA 37 °C No growth; MEA 20–21; MEA 30 °C 37–38; DG18 10–11; CYAS No growth; OA 35–37; CREA 5–9; YES 20–22.

Colony characters: CYA 25 °C, 7 days: Colonies raised at centre, slightly sulcate; margins low, plane, entire (2 mm); mycelium white; texture floccose; sporulation moderately dense, conidia en masse dull to greyish green (25D4–25D5); soluble pigments absent; exudates absent; reverse pale orange (5A3–6A3). MEA 25 °C, 7 days: Colonies raised, slightly sulcate; margins low, plane, entire (2 mm); mycelium white; texture floccose and velvety; sporulation dense, conidia en masse dull to greyish green (26D4–27D4); soluble pigments absent; exudates absent; reverse greyish orange (5B6) with reddish brown (8D8) dots and circle. YES 25 °C, 7 days: Colonies raised at centre, sulcate; margins low, plane, entire (2 mm); mycelium white; texture floccose; sporulation absent to sparse; soluble pigments absent; exudates absent; reverse brownish red (8C8) centre fading to light orange (5A4). DG18 25 °C, 7 days: Colonies sterile, raised, plane; margins low, plane, entire (2 mm); mycelium white; texture floccose; sporulation absent to sparse; soluble pigments absent; exudates clear droplets; reverse light orange (5A4). OA 25 °C, 7 days: Colonies low, plane; margins low, plane, entire (3–4 mm); mycelium white; texture velvety; sporulation moderately dense to dense, conidia en masse greyish green (26D5–26E5); soluble pigments absent; exudates absent; reverse pastel-red in centre fading to beige (at 30 °C violet red). CREA 25 °C, 7 days: weak growth, acid production absent.

Micromorphology: Conidiophores biverticillate; stipes smooth-walled, 125–450 × 2.2–3 μm; metulae divergent, 3–6, 8–13 × 2.5–4.5 μm; phialides ampulliform, tapering to a very thin neck, 3–6 per metula, 8.5–12 × 2.5–4 μm; conidia verrucose, rough-walled, globose, 2.5–4 × 2.5–4 μm.

Extrolites: Talaromyces francoae produces berkelic acid, mitorubrin, mitorubrinic acid and vermicillin.

Talaromyces purgamentorum Yilmaz, López-Quintero, Vasco-Pal. & Houbraken, sp. nov. Mycobank MB 816233. Fig. 5.

Etymology: named after de original substrate of the type strain, decomposed leaf litter debris.

Diagnosis: Conidiophores biverticillate with ampulliform phialides that taper to a very thin neck producing rough-walled, verrucose conidia. Lack of acid production on CREA and absence of growth on CYA at 37 °C. Restricted growth on YES (10–12 mm) and DG18 (5–6 mm) at 25 °C.

Typus: Colombia, dept. Amazonas, Peña Roja, ex mixed leaf litter from 4 months old litterbag in Pseudomonotes tropenbosii (Dipterocarpaceae) forest, isolated in March 2000, C. López-Quintero (holotype HUA 197222; culture ex-type CBS 113145 = IBT 23220 = DTO 056-E1).

ITS barcode: KX011504 (alternative markers: BenA = KX011487; CaM = KX011500).

Colony diameter, 7 days (mm): CYA 19–20; CYA 30 °C 24–25; CYA 37 °C No growth; MEA 20–22; MEA 30 °C 25–28; DG18 5–6; CYAS 2–3; OA 33–35; CREA 7–10; YES 10–12.

Colony characters: CYA 25 °C, 7 days: Colonies raised at centre, sunken at centre, sulcate; margins low, plane, entire (1 mm); mycelium white; texture loosely funiculose and velvety; sporulation dense, conidia en masse dull green (26D4–26E4); soluble pigments absent; exudates absent; reverse brown (6E7) centre reddish brown fading to greyish yellow (8D6 and 4B5). MEA 25 °C, 7 days: Colonies low, slightly sulcate; margins low, plane, entire (1–2 mm); mycelium white; texture velvety; sporulation dense, conidia en masse dull green (26D4–26E4); soluble pigments absent; exudates absent; reverse brownish orange (5C6–6C6). YES 25 °C, 7 days: Colonies slightly raised, sterile white aerial mycelium, slightly sulcate; margins low, plane, entire (<1 mm); mycelium white; texture floccose and funiculose; sporulation absent to sparse; soluble pigments absent; exudates absent; reverse bronze brown (5E5) centre fading to greyish orange (5B5). DG18 25 °C, 7 days: Colonies slightly raised, plane; margins low, plane, entire (<1 mm); mycelium white; texture velvety; sporulation dense, conidia en masse dull green (26D4–26E4); soluble pigments absent; exudates absent; reverse light brown (5D4–5D5). OA 25 °C, 7 days: Colonies low, plane; margins low, plane, entire (4–5 mm); mycelium white; texture velvety; sporulation moderately dense, conidia en masse dull green (28D4); soluble pigments absent; exudates absent; reverse pastel red. CREA 25 °C, 7 days: Weak growth, acid production absent.

Micromorphology: Conidiophores biverticillate; stipes smooth-walled, 170–300 × 2.8–4.2 μm; metulae divergent, 3–6, 7–11.5 × 3–4.5 μm; phialides ampulliform, tapering to a very thin neck, 3–6 per metulae, 7.5–12.5 × 3–4 μm; conidia rough-walled, globose to subglobose, 2–3.3 × 2–3 μm.

Extrolites: Talaromyces purgamentorum produces a dibenzofuran, penicillide, purpactin A, a shamixanthone and a violaceol. The dibenzofuran had the same UV spectrum as karnatakafurans A and B (Manniche et al. 2004), but was more apolar. Penicillide and purpactins (also called vermixocins) are related extrolites that have been isolated from several Talaromyces species, including T. derxii, T. purpurogenus and T. flavus (Sassa et al. 1974; Suzuki et al. 1991; Proksa et al. 1992). These compounds have very high levels of bioactivity, and are inhibitors of acyl-CoA:cholesterol acyltransferases and calpain (Tomoda et al. 1991; Chung et al. 1998).