Delimitation and characterisation of Talaromyces purpurogenus and related species

Taxa of the Talaromyces purpurogenus complex were studied using a polyphasic approach. ITS barcodes were used to show relationships between species of the T. purpurogenus complex and other Talaromyces species. RPB1, RPB2, β-tubulin and calmodulin sequences were used to delimit phylogenetic species in the complex. These data, combined with phenotypic characters, showed that the complex contains four species: T. purpurogenus, T. ruber comb. nov. and two new species T. amestolkiae sp. nov. and T. stollii sp. nov. The latter three species belong to the same clade and T. purpurogenus is located in a phylogenetic distant clade. The four species all share similar conidiophore morphologies, but can be distinguished by macromorphological characters. Talaromyces ruber has a very distinct colony texture on malt extract agar (MEA), produces bright yellow and red mycelium on yeast extract sucrose agar (YES) and does not produce acid on creatine sucrose agar (CREA). In contrast, T. amestolkiae and T. stollii produce acid on CREA. These two species can be differentiated by the slower growth rate of T. amestolkiae on CYA incubated at 36 °C. Furthermore, T. stollii produces soft synnemata-like structures in the centre of colonies on most media. Extrolite analysis confirms the distinction of four species in the T. purpurogenus complex. The red diffusing pigment in T. purpurogenus is a mixture of the azaphilone extrolites also found in Monascus species, including N-glutarylrubropunctamine and rubropunctatin. Talaromyces purpurogenus produced four different kinds of mycotoxins: rubratoxins, luteoskyrin, spiculisporic acid and rugulovasins and these mycotoxins were not detected in the other three species.

Both P. purpurogenum and P. rubrum are claimed to produce rubratoxins (Wilson & Wilson 1962, Moss et al. 1968, Natori et al. 1970). Because P. rubrum was not accepted by Pitt (1980) and P. purpurogenum has been regarded as a producer of glauconic acid rather than rubratoxins, Frisvad (1989) considered P. crateriforme to be the correct name for the species producing rubratoxins. Rubratoxin B is mutagenic, hepatotoxic, nephrotoxic and splenotoxic to several animals (Burnside et al. 1957, Lockard et al. 1981, Surjono et al. 1985, Engelhardt et al. 1987, Kihara et al. 2001). The first human rubratoxicosis was reported by Richer et al. (1997). Three teens drinking homemade rhubarb wine, which had a high level of rubratoxin B became critically ill, with one requiring immediate liver transplant. Even though rubratoxin B has negative health effects, it has potential as an anti-tumor agent (Wang et al. 2007, Wada et al. 2010. Penicillium crateriforme has also been reported to produce the mouse mycotoxin spiculisporic acid (Oxford & Raistrick 1934, Fujimoto et al. 1988). Later, spiculisporic acid has been used as a commercially available biosurfactant (Ishigami et al. 2000). Isolates belonging to P. crateriforme also produces the clavine alkaloids rugulovasines A and B and chlororugulovasines A & B (Dorner et al. 1980, the producer ATCC 44445 was identified as P. rubrum) (see Table 2). Penicillium purpurogenum is an important species in biotechnology for its ability to produce enzymes such as xylanases and cellulases (Steiner et al. 1994, Belancic et al. 1995 and pigments, which are used as natural colorants and biosorption (Say et al. 2004, Mapari et al. 2009, Jeya et al. 2010, Zou et al. 2012. Penicillium purpurogenum inoculated oak chips are used in artificial aging of Italian wines (Petruzzi et al. 2010(Petruzzi et al. , 2012   Identified as Penicillium purpurogenum var. rubisclerotiorum by Raper & Thom (1949). It produced limited numbers of dark red sclerotia. 3 The isolate was sent to CBS by S. Ochiai, Jonquil Consulting Inc., Tokyo, Japan. 4 Raper & Thom (1949) reported faster growth and floccose margins for this strain. Pitt (1980) does not mention this strain.

5
NRRL 1062 was used by Raper & Thom (1949) to describe Penicillium rubrum. 6 NRRL 1033 was identified as Penicillium funiculosum by Raper & Thom (1949 Benjamin (1955) introduced the name Talaromyces as a sexual morph and this genus was characterised as producing soft yellow ascomata that consist of interwoven hyphae. Following the concept of single name nomenclature, 40 species from Penicillium subg. Biverticillium were transferred and combined into Talaromyces ). The morphologically circumscribed species Penicillium purpurogenum sensu Pitt (1980) is one of several complexes of cryptic phylogenetic species that occur in the genus.
In the current study, the T. purpurogenus species complex was revised based on a polyphasic approach incorporating macroand micro-morphology, extrolite production and multi-gene derived phylogeny. The phylogenetic relationships between species of the T. purpurogenus complex and other members of Talaromyces are studied using ITS barcodes. For the detailed delimitation of phylogenetic species, sequences of four alternative genes, β-tubulin, calmodulin, RPB1 and RPB2, were used.

Strains
Cultures used for comparisons in this study were obtained from the culture collections of the CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands, the IBT culture collection, Lyngby, Denmark and fresh isolates deposited in the working collection of the Department of Applied and Industrial Mycology (DTO), housed at CBS. Strains studied are listed in Table 1.

Morphological analysis
Macroscopic characters were studied on 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 (Oxoid) (MEA). The strains were inoculated at three points on 90-mm Petri dishes and incubated for 7 d at 25 °C in darkness. All media were prepared as described by Samson et al. (2010). The temperature-growth response of strains was studied on CYA. Strains were inoculated at 3 points and incubated at 6,9,12,15,18,21,24,27,30,33,36 and 40 °C for 7 d in darkness. After incubation, the colony diameter on the various agar media was measured. The degree of sporulation, obverse and reverse colony colours and the production of soluble pigments were also determined. Colony colours were described using Kornerup & Wanscher (1967). Colonies were photographed with a Canon EOS 400D. Species were characterised microscopically by preparing slides from MEA. Lactid acid was used as mounting fluid. Specimens were examined using a Zeiss AxioSkop2 plus microscope, and the NIS-Elements D software package from Nikon was used for making photographs and taking measurements.

DNA extraction, PCR amplification and sequencing
DNA extractions were prepared from strains grown for 7 to 14 d on MEA using the Ultraclean TM Microbial DNA isolation Kit (Mo-Bio, Solana Beach, USA). Extracted DNA was stored at -20 °C. The ITS regions and regions of the β-tubulin, calmodulin, RPB1 and RPB2 genes were amplified and sequenced according to previously described methods (Houbraken et al. 2007).

Data analysis
Sequence contigs were assembled using Seqman from DNA-Star Inc. Newly generated ITS sequences were included in a dataset obtained from the Samson

RPB1 RPB2
β-tubulin calmodulin OA at 25 °C for 7 d for extrolite extraction. Three agar plugs of each medium were extracted as described in Nielsen et al. (2011) and Houbraken et al. (2012). The extracts were analysed by using high performance liquid chromatography with diode-array detection (HPLC-DAD) (Frisvad & Thrane 1987) for extracts made before 2011 and by UHPLC-DAD (Houbraken et al. 2012) for extracts made later. The compounds eluting and detected were identified by comparing retention time, retention index and UV spectra measured from 200 -600 nm. The UV spectra were compared to a database of UV spectra (Nielsen et al. 2011), and to literature data (see for example the UV spectrum of pestalasin A shown in Nonaka et al. 2011).

RESuLTS
Morphological examination of strains previously identified as T. purpurogenus showed the presence of four distinguishable morphological groups and these are treated here as distinct species ( Fig. 1): T. purpurogenus, T. ruber comb. nov., T. stollii sp.
nov. and T. amestolkiae sp. nov. Talaromyces purpurogenus is distinct from the other three species by its inability to grow below 18 °C, slow growth on the agar media CYA, the production of a bright red diffusing pigment on CYA at 25 °C and bright yellow and orange mycelium on DG18 at 25 °C. Talaromyces ruber has a velvety texture on both CYA and MEA at 25 °C and produces bright yellow and red mycelium on YES. It also produces a very distinct colony texture on MEA, where bundles of hyphae are produced underneath the velvety texture. Talaromyces amestolkiae and T. stollii are distinguished from T. ruber and T. purpurogenus by the production of acid on CREA. Talaromyces stollii, however, does grow faster on CYA at 36 °C than T. amestolkiae and some of the studied T. amestolkiae strains produced sclerotia after 2 wk incubation at 25 °C. Furthermore, T. stollii has soft synnemata-like or tufted structures at the centre of colonies on most media. Morphological data is supported by phylogenetic results, as discussed below (Fig. 2, 3).
Barcodes of the ITS locus were used to study the phylo genetic relationship between strains previously identified as T. purpuro-   Pitt (1980), is distinct from T. minioluteus (CBS 642.68 T ) and resolved in the T. ruber clade (Fig. 2). Many strains previously identified as Penicillium purpurogenum var. rubrisclerotium were resolved in a clade with T. amestolkiae. However, the ex-type strain of P. purpurogenum var. rubrisclerotium (CBS 270.35 T ) is resolved in a distinct clade closely related to T. minioluteus (Fig. 2).

dISCuSSIon
Cultures that previously were identified as P. purpurogenum or P. rubrum were analysed in this study and phylogenetic, morphological and extrolite results show that the T. purpurogenus complex consists of four distinct species. The species described below are quite common on textiles, paper, soil, dung, plant debris, coffee-berries, corn, indoor air and dust, and are distributed worldwide. Talaromyces purpurogenus has been implicated in the biodeterioration of cellulose materials such as textiles, paper and adhesives, while it also has the ability to grow on plant material such as corn, where it may produce mycotoxins (Moss et al. 1971). Talaromyces purpurogenus produces four types of mycotoxins: rubratoxin A & B, rugulovasins, spiculisporic acid and luteoskyrin, and none of the other three species treated have been found to produce mycotoxins. The newly described species T. amestolkiae and T. stollii grow well at 37 °C and some strains were isolated from AIDS patients and might be opportunistic pathogens. It is not yet known if any other species in this group can be opportunistic pathogens. Talaromyces purpurogenus was reported as the causal agent of a disseminated mycosis in a German shepherd dog (Zanatta et al. 2006), but it remains unknown if this species identification is correct using the newly proposed taxonomy. This group is also biotechnologically important, because of their production of enzymes (Carvallo et al. 2003, Jeya et al. 2010 and extrolites. For example, the mycotoxin rubratoxin A & B produced by T. purpurogenus has been shown to act as cancer metastasis suppressors (Wada et al. 2010) and spiculisporic acid can be used as a detergent (Ishigami et al. 2000). From a biotechnological point of view we would recommend using T. ruber for enzyme production, because T. purpurogenus produces four types of mycotoxins and T. amestolkiae and T. stollii are potentially pathogenic to immuno-compromised persons. However, it is not known whether the enzymes reported from T. purpurogenus (Steiner et al. 1994, Belancic et al. 1995 are indeed from this species or one of the other three taxa treated here or even any of them. Most of the isolates produced the extrolites characteristic of the species (Table 3), but some isolates should be grown on other media to examine whether they can also produce the remaining extrolites found in productive strains. Most extrolites supported the phylogram in Table 3. Production of purpactin, pestalasin A, vermicellin and 'm334' supported that T. ruber and T. amestolkiae are closely related. On the other hand common production of 'HHH' indicated that T. amestolkiae and T. stollii are closely related, while common production of austin indicates that T. ruber and T. stollii are closely related. Purpactin was pro-exudate absent, in some strains clear; soluble pigment absent; reverse coloration reddish brown (8D7). DG18, 25 °C, 7 d: Colonies 14 -16 mm, plane, low, with a brownish orange colour; margins narrow (2-3 mm), entire; mycelium white; texture floccose; sporulation sparse, conidia en masse similar to CYA; exudate small clear droplets; soluble pigment absent; reverse coloration greyish green (30D6 -30E6) at centre, elsewhere greenish white (30A2). YES, 25 °C, 7 d: Colonies 22 -30 mm, low, raised at centre, radially and concentrically sulcate; margins low, narrow (1-2 mm), entire; mycelium white and yellow, red in some strains, e.g. CBS 868.96; texture floccose; sporulation sparse to moderate dense, conidia en masse greyish green (27C5 -27E6 -27E7); exudate clear small droplets; reverse coloration greyish brown to brown (5F8 -5F3) near centre, at margins brownish orange to light brown (5C4 -5D4). CREA, 25 °C, 7 d: Colonies 10 -14 mm, restricted growth, no acid production. CYAS, 25 °C, 7 d: Typically no growth, sometimes microcolonies up to 4 mm.
Distinguishing characteristics -Talaromyces ruber can be distinguished from T. purpurogenus by growth at lower temperatures, having a velvety texture on MEA, yellow mycelia and bright green conidia on YES after 7 d incubation at 25 °C. Talaromyces ruber can be distinguished from T. stollii and T. amestolkiae by absence of acid production on CREA. Talaromyces ruber has a velvety structure on both CYA and MEA at 25 °C, produces a very distinct colony texture on MEA and produces bright yellow and red mycelia on YES. Yilmaz, Houbraken, Frisvad & Samson, sp. nov. -MycoBank MB801359;Fig. 7 Etymology. Latin, stollii: named in honour of Otto Stoll, a pharmacist who first described P. rubrum and P. purpurogenum for his PhD thesis at the K. Bayr Julius Maximilians University in Würzburg, Germany in 1905.
The species in this complex generally produce yellow, orange and red pigments in the mycelium or as diffusing pigments. Extro lites responsible for these colours are two groups of azaphilone polyketide pigments the mitorubrins (mitorubrin, mitorubrinol, mitorubrinol acetate and mitorubrinic acid) (Büchi et al. 1965) and the Monascus red pigments (N-glutaryl monascorubramin, N-glutarylrubropunctamin, monascorubramine, monascin, PP-R and others (Mapari et al. 2009)). These azaphilone polyketides are produced by all the species treated in this paper and several other species in Talaromyces, but they appear to be produced in different ratios and amounts in different isolates and species , van Reenen-Hoekstra et al. 1990. Also, especially on MEA, we observed that when a strain that produced red pigment was transferred to another MEA plate, the strain sometimes lost the ability to produce the red pigment. However, red pigment production was consistent on CYA. Apart from the medium employed for extrolite production, the age of the strain may also play a role: older strains of T. purpurogenus, such as isolates formerly called P. crateriforme and P. sanguineum, have lost their ability to produce high amounts of diffusible red pigments. The red pigments have resulted in some confusion, especially in the concept of T. purpurogenus and T. ruber. Talaromyces purpurogenus and T. ruber were described by Stoll (1903Stoll ( -1904. Raper & Thom (1949) considered the species as distinct. Talaromyces purpurogenus was distinguished from T. ruber by the production of spreading dark yellow green colonies and smooth-walled conidia in the latter species. This is in comparison to the sometimes more restricted dark green colonies and rough-walled conidia they observed in T. purpurogenus. Although Pitt (1980) synonymised T. ruber with T. purpurogenus, our data indicate that these two species are distinct and they are re-described below. Talaromyces ruber can be distinguished from T. purpurogenus by growth at lower temperatures, its velvety texture on MEA, yellow mycelium and bright green conidia on YES after 7 d incubation at 25 °C. With regards to conidia ornamentation, all strains examined of both these species produced smooth-walled conidia and this character is thus not diagnostic for species recognition. No type material was designated for T. ruber, therefore Raper & Thom (1949) centred their description of T. ruber on NRRL 1062 and NRRL 2120. Our analysis shows these two strains belong to different species. NRRL 1062 (= CBS 370.48) is designated here as the neotype of T. ruber, while NRRL 2120 represents a new phylogenetically unrelated species (Fig. 2).
Penicillium sanguineum and P. crateriforme are considered synonyms of T. purpurogenus. In Sopp's description of P. sanguineum, he states that this species produces bright red pigments, which colours the entire gelatine medium, as well as producing yellow coloured mycelium (Sopp 1912). Although no type material exist for this species, the description by Sopp (1912) indicates that it belongs to the T. purpurogenus complex. Penicillium crateriforme (CBS 184.27 T ) is resolved in a clade together with the ex-type cultures for T. purpurogenus (CBS 286.36 T) and is considered a synonym of T. purpurogenus. Pitt (1980) neotypified P. minioluteum using strain IMI 89377ii (CBS 196.88). CBS 642.68 T is a subculture of the same strain obtained from the IMI in 1968, but it morphologically fits Biourge's description of P. minioluteum. It was therefore considered the correct neotype of the species as discussed in earlier studies (van Reenen-Hoekstra et al. 1990). Our phylogenetic data show that T. minioluteus (CBS 642.68) remains in a clade distantly related to T. ruber (CBS 196.88).
This study resulted in the delimitation of T. amestolkiae and T. stollii, two new species closely related to T. ruber. Talaromyces amestolkiae and T. stollii are distinguished from T. purpurogenus and T. ruber by their acid production on CREA and floccose to funiculose texture of MEA. Compared to T. amestolkiae, T. stollii grows faster on CYA at 36 °C, as well as producing unique synnemata/tufted mycelium on most media. Talaromyces amestolkiae and T. stollii share the production of the 'HHH' family of extrolites. Although these species are resolved amongst known sexual species, we did not observe cleistothecia for strains studied. Future studies that aim to induce sexual reproduction would be interesting, especially for explaining the morphological and genetic variation observed between T. stollii strains. Also, sclerotia were produced by T. amestolkiae strains, but these never matured into cleistothecia. Many strains previously identified as P. purpurogenum var. rubrisclerotium were resolved in a clade with T. amestolkiae. However, the ex-type strain of P. purpurogenum var. rubrisclerotium (CBS 270.35 T ) is resolved in a distinct clade closely related to T. minioluteus ).
This paper addressed the taxonomic difficulties experienced in the T. purpurogenus complex. Results showed that this complex contains four distinct species and that they can be identified using morphological characters, extrolites and/or genetic data. The ITS barcodes could reliably separate the four species within this complex. However there is only one base pair difference between T. ruber and T. amestolkiae, and thus the alternative genes were needed for taxon identification. Although calmodulin could not resolve T. amestolkiae from T. ruber, RPB1, RPB2 and β-tubulin gave a clear species delineation and can be used for identifying species within this clade.