Two new Penicillium species Penicillium buchwaldii and Penicillium spathulatum, producing the anticancer compound asperphenamate


Correspondence: Jens C. Frisvad, Department of Systems Biology, Center for Microbial Biotechnology, Søltofts Plads, Building 221, DK-2800 Kgs. Lyngby, Denmark. Tel.: +4545252626; fax: +4545884922; e-mail:


Penicillium buchwaldii sp. nov. (type strain CBS 117181T = IBT 6005T = IMI 30428T) and Penicillium spathulatum sp. nov. (CBS 117192T = IBT 22220T) are described as new species based on a polyphasic taxonomic approach. Isolates of P. buchwaldii typically have terverticillate conidiophores with echinulate thick-walled conidia and produce the extrolites asperphenamate, citreoisocoumarin, communesin A and B, asperentin and 5′-hydroxy-asperentin. Penicillium spathulatum is unique in having restricted colonies on Czapek yeast agar (CYA) with an olive grey reverse, good growth on CYA supplemented with 5% NaCl, terverticillate bi- and ter-ramulate conidiophores and consistently produces the extrolites benzomalvin A and D and asperphenamate. The two new species belong to Penicillium section Brevicompacta and are phylogenetically closely related to Penicillium tularense. With exception of Penicillium fennelliae, asperphenamate is also produced by all other species in section Brevicompacta (P. tularense, Penicillium brevicompactum, Penicillium bialowiezense, Penicillium olsonii, Penicillium astrolabium and Penicillium neocrassum). Both new species have a worldwide distribution. The new species were mainly isolated from indoor environments and food and feedstuffs. The fact that asperphenamate has been found in many widely different plants may indicate that endophytic fungi rather than the plants are the actual producers.


Asperphenamate (= asjanin = anabellamide = auranamide) was first discovered from Aspergillus flavipes by Clark et al. (1977) and Clark & Hufford (1978) and later reported from Penicillium brevicompactum (Doerfler et al., 1981; Bird & Campbell, 1982a, b; Bringmann et al., 2010; Frisvad et al., 2004), Penicillium bialowiezense (Frisvad et al., 2004), Penicillium olsonii (Frisvad et al., 2004), Penicillium megasporum (Nozawa et al., 1989), Penicillium soppii (Frisvad et al., 2006), Penicillium canadenseP. arenicola = Phialomyces arenicola (McCorkindale et al., 1978; Houbraken & Samson, 2011), Aspergillus janus (Nakashima et al., 1983; as asjanin), Aspergillus allahabadii, Aspergillus carneus and Aspergillus microcysticus (Samson et al., 2011) and Talaromyces thailandense (Dethoup et al., 2007). These species all belong to the Trichocomaceae (Houbraken & Samson, 2011). However, asperphenamate has also been found in a series of unrelated plant species: Anaphalis subumbellata (Compositae), Artemisia anomala (Asteraceae), Begonia nantoensis (Begoniaceae), Cantharanthus pusillus (Apocunaceae), Croton hieronymi (Euphorbiaceae), Desmos longiflorus (Annonaceae), Dorstenia dinklagei (Moraceae), Ficus mucoso (Moraceae), Grangea maderaspatana (Compositae), Leucas aspera (Lamiaceae), Medicago polymorpha (Fabaceae), Melastroma malabathricum (Lamiaceae), Miliusa velutina (Annonaceae), Piper aurantiacum (Piperaceae), Piptadenia gonoacantha (Leguminosae), Saurauia napaulensis (Actinidiaceae), Uvaria ufa (Annonaceae), Wikstroemia indica (Thymelaceae) and Zeyhera digitalis (Bignoniaceae) (Battersby & Kapil, 1965; Banerji & Ray, 1981; Wu et al., 2004; Talapatra et al., 1983; Poi & Anityachoudhury, 1986; Jakupovic et al., 1987; Singh & Jain, 1990; Catalán et al., 2003; Bankeu et al., 2010; Geng et al., 2006; Pomini et al., 2006; Sandhu et al., 2006; Vouffo et al., 2008; Xiao et al., 2007; Carvalho et al., 2010; Macabeo et al., 2010; Sirat et al., 2010) leading to the suggestion that asperphenamate may be produced by endophytic fungi rather than plants (Macabeo et al., 2010). Asperphenamate has recently attracted much interest because of its antitumor (Wu et al., 2004; Yuan et al., 2010, 2012; Li et al., 2012) and antimicrobial activity. Furthermore, filamentous fungi are more sustainable and efficient industrial producers of secondary metabolites than the plants, so there is interest in finding further, perhaps more efficient producers of asperphenamate among the fungi. In our studies, on the biodiversity of Penicillia, we found a large number of isolates producing a rich profile of extrolites including asperphenamate. In addition, we found that these strains grow well at lower temperatures and are halotolerant.

Growth in cold habitats often involves the ability to grow at low water activities because liquid water is only partially available in ice and maybe xerophile plays a role in psychrophily or psychrotolerance (Petrovič et al., 2000; Gunde-Cimerman et al., 2003). In contrast to Aspergillus and its teleomorphs, no genuinely halotolerant species have been described in Penicillium. However, several species do grow better on Czapek yeast agar (CYA) with 5% NaCl than without (Frisvad & Samson, 2004; Frisvad, 2005; Houbraken et al., 2011). Many Penicillium species grow just as well or slightly slower at 15 °C than at 25 °C. The number of Penicillium species that grow better at 15 °C than at 25 °C is more limited, and these include Penicillium thymicola, Penicillium verrucosum, Penicillium marinum, Penicillium nothofagi and Penicillium wellingtonense (Frisvad & Samson, 2004; Houbraken et al., 2011). Until now, only Penicillium jamesonlandense has been described as psychrotolerant, and this species grows slowly or not at all at 25 °C and this thus is close to being psychrophilic (Frisvad et al., 2006).

We have encountered a large number of isolates that grow very slowly on CYA and other media at 25 °C but that grow significantly faster either at 15 °C or with 5% NaCl in the growth medium. A related series of similar Penicillium isolates were isolated from air and soil, but these were less halo- and psychrotolerant. These two groups of Penicillia were shown to be closely related, both concerning their phenotypes and their phylogenetic placement and appeared to be new species. The new taxa had some resemblance to Penicillium section Brevicompacta including P. brevicompactum, P. bialowiezense (= P. biourgeianum), P. olsonii, Penicillium astrolabium and Penicillium neocrassum (Frisvad & Samson, 2004; Peterson, 2004; Serra & Peterson, 2007) but also to Penicillium section Ramosa (Stolk & Samson, 1985; Houbraken & Samson, 2011). The two new taxa are described here using a polyphasic taxonomic approach using molecular data (RPB2, ITS, partial β-tubulin and calmodulin sequences), morphology, phenotypic cultural characters, physiological features and extrolite profiles.

Material and methods


The isolates examined (Table 1) were obtained from different culture collections or isolated from soil, food or feed samples on the selective medium DG18 (for formula see Samson et al., 2010). Thirty-nine strains of Penicillium buchwaldii were examined (Table 1) of which 15 were analysed chemically. Sixty-four strains of Penicillium spathulatum were examined (Table 1) of which 19 were examined chemically. In addition, species belonging to section Brevicompacta (Penicillium tularense, 21 strains) (Table 1) of which 20 strains were examined chemically, P. brevicompactum, P. bialowiezense and P. olsonii (data in Frisvad et al., 2004), P. astrolabium (NRRL 35611 = IBT 28865) and P. neocrassum (NRRL 35639 = IBT 28863; NRRL 35648 = IBT 28868) were examined for extrolites. The cultures were maintained in the culture collections of the DTU, Department of Systems Biology, Denmark (IBT), and selected isolates were accessioned at the culture collection of CBS-KNAW Fungal Biodiversity Centre, the Netherlands.

Table 1. Penicillium isolates used in this study
SpeciesIBT collection numberOther culture collection numbersSourceGeographic origin, isolator, year
P. tularenseIBT 4901 = IBT 18422 = IBT 24559CBS 430.69 = IMI 148394 = FRR 899 = ATCC 22056 (ex type)Soil under Pinus ponderosa and Quercus kelloggiiPine Flat, Tulane county, California, USA, J.W. Paden, 1968
IBT 12741WSF 2084 = CBS 126811A1 horizon soil, Floodplain, maple-elm-ash forestS. Wisconsin, M. Christensen 1960–1961 (as P. brevicompactum)
IBT 13144WSF 5840Soil, willow-cottonwood forestWisconsin, M. Christensen, 1962 (as P. stoloniferum)
IBT 13157 = IBT 24474RMF S36 = CBS 131234Soil under Artemisia tridentataRock Springs, Wyoming, M. Christensen, 1978
IBT 13180WSF 2222A1 horizon soil, Floodplain, maple-elm-ash forestS. Wisconsin, M. Christensen 1960–1961 (as P. brevicompactum)
IBT 14066RMF 8830Soil, native deciduous forestCoweeta LTER, M. Christensen
IBT 14705 = IBT 14789CBS 431.69 = FRR 913Soil under Pinus ponderosa and Quercus kelloggiiPine Flat, Tulane county, California, USA, J.W. Paden, 1968
IBT 16624 and IBT 16626 Soil under Populus angustifoliaFort Steele, near North Platte River, Wyoming, USA, J.C. Frisvad, October 1994
IBT 18139 Soil under Pinyon pineOwl Canyon, Livermore, Colorado, USA, J.C. Frisvad, November 1994
IBT 18423 = IBT 21632CBS 432.69Forest soilCalifornia, USA, J.W. Paden, 1968
IBT 18877, IBT 19095 and IBT 19107 Air in cake factoryGive, Denmark, J.C. Frisvad, 1996
IBT 21837 Colony on Pleurotus osteatusDenmark, J.C. Frisvad
IBT 22768 Tundra soil under Erigeron sp. and Krumholz pineLibby Flats, Snowy Range, Wyoming, J.C. Frisvad, July 1996
IBT 22729 Tundra soil under Geum rossiiLibby Flats, Snowy Range, Wyoming, J.C. Frisvad, July 1996
IBT 22768 Tundra soilLibby Flats, Snowy Range, Wyoming, J.C. Frisvad, July 1996
IBT 26476CBS 116045Air in cardboard factoryNetherlands, J. Houbraken, 2004
IBT 27015 Soil under leaves of Coffea Arabica (Coffee plantation)Netrakonda, near Chickmagalur, India, December 1996
Isolate tom1 and tom2 Mouldy tomatoDenmark, B. Andersen (Andersen & Frisvad, 2004)
Penicillium buchwaldiiIBT 3748 = IBT 3749 = IBT 6006IMI 304288Contaminant on Petri dishKongens Lyngby, Denmark, J.C. Frisvad, 1983
IBT 3750 Wheat from a suspected case of mycotoxicosis in pigs 
IBT 3751IMI 304289Linum usitatissimumKongens Lyngby, Denmark, J.C. Frisvad, June, 1984
IBT 3752CBS 116935WheatUnited Kingdom, K.A. Scudamore and J.H. Clarke
IBT 6003 Soil 
IBT 6004IMI 304287Hordeum vulgareØrsted, Denmark, J.C. Frisvad, June 1981
IBT 6005CBS 117181 = IMI 304286 (ex type)Hordeum vulgareØrsted, Denmark, J.C. Frisvad, June 1981
IBT 6007 SoilHoutrijbdijk, Netherlands, J.C. Frisvad
IBT 6747 Wheat flourDenmark, J.C. Frisvad
IBT 6748 Unknown sourceDenmark
IBT 6843 Unknown sourceDenmark
IBT 10121 Millet imported to DenmarkSenegal, J.C. Frisvad, 1990
IBT 12084 and IBT 12086 Margarine containerDenmark
IBT 12130 Air in factoryDenmark
IBT 15345CBS 116980WheatUnited Kingdom, K.A. Scudamore and J.H. Clarke
IBT 15335 and IBT 15346 WheatUnited Kingdom, K.A. Scudamore and J.H. Clarke
IBT 15430CBS 116931WheatUnited Kingdom, K.A. Scudamore and J.H. Clarke
IBT 15740 Air in factoryDenmark
IBT 15909CBS 116932Air, ceilingDenmark
IBT 16812CBS 116934YoghurtDenmark
IBT 17826IMI 101414Unknown source 
IBT 20032 Air in cake factoryDenmark
IBT 21422CBS 116929Wheat flourDenmark
IBT 21980CBS 116930Indoor airDenmark, C.K. Wilkins
IBT 22577 and IBT 22578 Wheat flourBari, Italy
IBT 22579CBS 116933Foccacia breadFiorenza, Bari, Italy
IBT 26521 Quercus suber leafPenas Blancas (N 36° 26.772′. W 5° 14.942) 1137 feet above sea level, Spain, November 2004, B. Andersen and J.C. Frisvad
IBT 27223 Air in dairyOdense, Denmark
IBT 28159 and IBT 28160 StrawberryFrederiksberg, Copenhagen, Denmark
IBT 30016 Soil under Chamaenerion latifoliumNuuk, Greenland, E.K. Lyhne, 2006
IBT 31137EXF 5592SalternSlovenia, Nina Gunde Diderichsen
IBT 31226 Unknown source 
IBT 31631 Unknown source 
Isolate AS-JCF-17B-BV12 Air in bread factoryDenmark
 Isolate D.70Rum3 Air in factoryDenmark
P. spathulatumIBT 5921 = IBT 6469 Poa annuaDenmark
IBT 6008 Wheat field soilFlakkebjerg, Denmark, S. Elmholt, 1986
IBT 6009 Soil near lakesideLoosdrecht, the Netherlands, J.C. Frisvad, 1987
IBT 6291 SoilImperial Garden, east, Tokyo, J.C. Frisvad, August 1988
IBT 12083 Margarine containerDenmark
IBT 12357, IBT 12441, IBT 12448, IBT 12617, IBT 12619 - IBT 12626 SalamiDenmark
IBT 12670 Kangaroo ratSevilletta National Wildlife Refuge, Socorro County, New Mexico, USA, L. Hawkins
IBT 12671 Buried seedSevilletta National Wildlife Refuge, Socorro County, New Mexico, USA, L. Hawkins
IBT 13038CBS 327.92Green mouldy goat cheeseCrete, Greece, P.V. Nielsen
IBT 13075 PearUSA, 1992
IBT 13260 Mouldy cheeseDenmark, J.C. Frisvad
IBT 14168 Rye breadDenmark
IBT 14188 Wheat flourDenmark
IBT 14211 SoilEastern Island, Chile, 1990
IBT 16589 Soil under sage brushAlong Buffalo Fork River, 7 miles east of Moran Junction, Wyoming, M. Christensen and J.C. Frisvad, USA, October 1994
IBT 16777 Sesame seedsImported to Denmark, 1995
IBT 16778 Air, bathroomDenmark
IBT 16779 Lotus rootJapan, 1988
IBT 18526 and IBT 18787 Air, cake factoryDenmark
IBT 18983CBS 116976Chalky soilFaxe limestone quarry, Sealand, Denmark, S. Banke
IBT 19530CBS 116977Chalky soilFaxe limestone quarry, Sealand, Denmark, S. Banke
IBT 21021 Unknown source 
IBT 22220CBS 117192 (ex type)Mouldy chestnut (Castanea sp.)Imported from France to Denmark, P.V. Nielsen
IBT 22248 Soil under Raphia palm in primary forestLas Alturas, elev. 1550 m, Costa Rica, D. Tuthill
IBT 22413CBS 116975Soil under Raphia palm in primary forestLas Alturas, elev. 1550 m, Costa Rica, D. Tuthill
IBT 22466CBS 116974Soil under Pinus sp.Chile, D. Tuthill
IBT 22480CBS 116973Soil under Nothofagus sp.Chile, D. Tuthill
IBT 22530 Dried whale meatScoresbysund, Greenland, A. Lynge
IBT 22640CBS 116972Sandy soilFortune Bay, Disco Island, Greenland; S. Gravesen
IBT 24432 ChestnutDenmark
IBT 24436 Dried whale meatScoresbysund, Greenland, A. Lynge
IBT 26219 Tulipa bulbDenmark
 IBT 26401, IBT 26420, IBT 26421, IBT 26425, IBT 26427 and IBT 26478 Rainwater from cover of tent, camp near inland icePaakitsoq, Greenland, N69° 26.168′, W 50° 16.569′, E.K. Lyhne, August 2004
IBT 26450 Inland icePaakitsoq, Greenland, N69° 25.830′, W 50° 15.199′, E.K. Lyhne, August 2004
IBT 26479CBS 115987MargarineNetherlands, J. Houbraken
IBT 26758 Soil, coffee plantationChikmagalur, India
IBT 27987 and IBT 27999 Sea water near beachBellevue, Denmark
Isolate JOSC7, JOSC11, JOSC14 and JOSC17 Soil under water millAgnetaryd, Sweden, J.C. Frisvad, July 1985
Isolate Joha 8 SoilHven, Sweden
Isolate SE 143 Wheat field soilFlakkebjerg, Denmark, S. Elmholt, 1986
Isolate JSDB 2 SoilSandbjerg, Denmark, J.C. Frisvad, October 1986
Isolate IDH222S5 CarrotKolding, I.D. Hansen, Denmark, 1988
Isolate ASJSL1242Y Liver pateÅbenrå, Denmark

Morphological examination

The isolates were inoculated on CYA, malt extract agar (MEA; Oxoid), creatine sucrose agar (CREA), yeast extract sucrose agar (YES) and CYA supplemented with 5% NaCl. Medium compositions follow Samson et al. (2010). In addition, the isolates were inoculated onto CYA and incubated at 6, 9, 12, 15, 18, 21, 24, 27, 30, 33 and 37 °C in darkness for 7 days. For micro-morphological observations, mounts were made in lactic acid from colonies grown on MEA, and a drop of alcohol was added to remove air bubbles and excess conidia.

Extrolite analysis

Cultures were grown on the agar media CYA and YES for 7 days at 25 °C prior to extraction. Extrolites were analysed by HPLC using alkylphenone retention indices and diode array UV–VIS detection as described by Houbraken et al. (2012). Standards of asperphenamate, asperentin, 5′-hydroxyasperentin, communesin A, communesin B, citreoisocoumarin, cyclopenol, cyclopenin, cyclopeptin and other extrolites from the collection at DTU Department of Systems Biology (Denmark) were used to confirm the identification of the extrolites from the species under study (Nielsen et al., 2011).

Isolation and analysis of nucleic acids

DNA was extracted and stored as described by Houbraken et al. (2011). Fragments containing the ITS regions (including the 5.8S) and a part of the 28S rDNA were amplified using primers V9G and LS266, as described previously by Van den Ende & de Hoog (1999). Amplification of part of the β-tubulin gene was performed using the primers Bt2a and Bt2b (Glass & Donaldson, 1995), and amplification of a part of the calmodulin gene was set up as described by Houbraken et al. (2007). A part of the RNA polymerase gene (RPB2) was amplified as described by Houbraken et al. (2012). Sequence analysis was performed with a Big Dye Terminator Cycle Sequencing Ready Reaction kit for both strands, and the sequences were aligned with the MT Navigator Software (Applied Biosystems). The resulting sequences of all the isolates were aligned using the muscle software implemented in mega5 package (Edgar, 2004; Tamura et al., 2011).

Analysis of sequence data

Four different loci were sequenced, and each data set was subjected to a maximum likelihood (ML) analysis using the mega5 software. The robustness of trees in the analyses was evaluated by 1000 bootstrap replicates. To further investigate the robustness of the phylograms, a Bayesian tree inference analysis using mrbayes v3.1.2 (Ronquist & Huelsenbeck, 2003) was performed. Prior to analysis, the most suitable substitution model was determined using mrmodeltest 2.3 (Nylander 2004), utilizing the Akaike information criterion. The RPB2 data set was used to determine the phylogenetic placement of the new species within Penicillium, and this phylogram was rooted with Talaromyces flavus (CBS 310.38). The calmodulin, ITS and β-tubulin data sets were analysed separately to determine whether the clades can be recognized as independent evolutionary lineages, but were also combined, to resolve the relationships among the examined strains. The phylograms were rooted with Penicillium canescens NRRL 910. Newly obtained sequences were deposited in GenBank under accession numbers JX313134JX313185. For phylogenetic analyses, data sets were supplemented with sequences from the study of Serra & Peterson (2007) and Samson et al., (2004).


Morphology and extrolites

Two new species were present among the examined strains, and we propose to name them Penicillium buchwaldii sp. nov. and Penicillium spathulatum sp. nov. Each species produced a unique extrolite pattern, and all isolates of each species had similar micro- and macromorphology and physiological characters. Detailed descriptions are given in the Taxonomy section of this manuscript. All P. buchwaldii isolates produced asperphenamate, asperentin, 5′-hydroxyasperentin, Raistrick phenols, communesin B and 13 of 15 isolates produced citreoisocoumarin (Table 2). All isolates of Pspathulatum produced asperphenamate and perinadine (reported from a strain of Penicillium citrinum see Sasaki et al., 2005), and 17 of the 19 isolates produced benzomalvins. Five of the 19 isolates produced cyclopenol and related benzodiazepins, and 5 of 19 produced breviones. Two isolates produced quinolactacin and 8 of 19 of the isolates produced indol alkaloids with an unknown structure. All chemically examined P. tularense isolates (20) produced paxillins, janthitrems and asperphenamate, except three strains that did not produce asperphenamate (CBS 431.69, CBS 432.69 and IBT 14066). Few isolates produced ascomata, and the growth rates of this species were in general similar to those of P. spathulatum.

Table 2. Production of extrolites by strains of Penicillium buchwaldii, Penicillium spathulatum and Penicillium tularense
P. buchwaldiiAsperentin15/15
Raistrick phenols15/15
Communesin A11/15
Communesin B15/15
P. spathulatumAsperphanamate19/19
Cyclopeptin, cyclopenin, and/or cyclopenol5/19
Specific indol alkaloids8/19
P. tularenseAsperphenamate17/20
Paxillin and derivatives20/20

Examination of P. astrolabium, P. neocrassum, P. brevicompactum, P. bialowiezense, Penicillium fennelliae and P. olsonii showed that all these species, except P. fennelliae, produced asperphenamate, and three of these species were able to produce breviones (P. bialowiezense, P. neocrassum NRRL 35648 and P. olsonii). Although the new species and members of section Brevicompacta share extrolites, they differ in their penicillus branching. Most species in section Brevicompacta have appressed rami, while P. spathulatum and P. buchwaldii have 2 or 3 divergent rami, similar to Penicillium lanosum, P. soppii and Penicillium scabrosum and other species in the sister clade to Brevicompacta, section Ramosa. Phenotypically, P. soppii in section Ramosa has two kinds of extrolites in common with the two new species (Frisvad et al., 2006), sharing asperphenamate and benzomalvins with P. spathulatum and asperphenamate with P. buchwaldii.

Phylogenetic analysis

Partial RPB2 sequences were used to determine the phylogenetic position of P. buchwaldii and P. spathulatum. The analysis involved 51 nucleotide sequences, and the length of the aligned RPB2 data set was 914 nucleotides. The phylogram based on the RPB2 data (Fig. 1) shows that the type species of section Ramosa (P. lanosum) and section Brevicompacta (P. olsonii) are together on a highly supported branch [100% bootstrap support (bs), 1.00 posterior probability (pp)]. Bayesian analysis shows that the species belonging to section Brevicompacta are together in a clade (1.00 pp); however, this branch lacks statistical support in the ML analysis (< 70%). Penicillium buchwaldii and P. spathulatum are closely related and in the same clade as members of section Brevicompacta (Fig. 1).

Figure 1.

Phylogenetic tree based on the RPB2 data set. The bootstrap values of the ML and posterior probability values of the Bayesian analysis are presented at the nodes (bs/pp). Only values above 70% bs and 0.95 pp are shown, and those with full support are indicated with an asterisk. If the bootstrap support was higher than 95%, then the internal branch is shown as a thicker line. Talaromyces flavus (CBS 310.38) is used as an outgroup.

The ITS regions and parts of the calmodulin and β-tubulin gene were sequenced to determine the phylogenetic relationship among members of section Brevicompacta. The length of the ITS, calmodulin and β-tubulin data sets were 679, 406 and 378 bp long, respectively. The topology of the phylograms of the three loci was generally similar (Fig. 2). In all three loci, P. buchwaldii and P. spathulatum are closely related to each other (> 80% bs; 1.00 pp). The main difference was the position of P. tularense. In the ITS phylogram, this species is in a clade together with P. buchwaldii and P. spathulatum (82% bs, 1.00 pp), while no statistical support was found for this position in the other two data sets. Based on the combined data set, P. buchwaldii and P. spathulatum are closely related, with P. tularense as a sister species (81% bs, 1.00 pp) (Fig. 2).

Figure 2.

Phylogenetic tree calculated from combined ITS regions (incl. 5.8S and partial 28S rDNA), calmodulin and β-tubulin data. The bootstrap values of the ML and posterior probability values of the Bayesian analysis are presented at the nodes (bs/pp). Only values above 70% bs and 0.95 pp are shown, and those with full support are indicated with an asterisk. If the bootstrap support was higher than 95%, then the internal branch is shown as a thicker line. Penicillium canescens NRRL 35656 is used as an outgroup.


Penicillium buchwaldii and P. spathulatum are most closely related to P. tularense, P. brevicompactum, P. bialowiezense, P. neocrassum and P. astrolabium and should therefore be classified in Penicillium section Brevicompacta (Houbraken & Samson, 2011). The close phylogenetic relationship between P. buchwaldii and P. spathulatum is reflected in certain extrolite similarities, and the phylogenetic relationship to P. tularense is also supported by similarities in penicillus structure and growth rates. Furthermore, P. tularense produces asperphenamate, paspaline, paspalinine, paxillin, dihydropaxillin and janthitrems (Andersen & Frisvad, 2004, results obtained here) and thus shares asperphenamate with P. buchwaldii and P. spathulatum. However, with exception of P. fennelliae, this extrolite is also produced by the other species in section Brevicompacta.

Penicillium spathulatum differs from other species in section Brevicompacta by growing slowly without NaCl or growing very restrictedly at 25 °C but faster in the presence of NaCl or lower temperatures (Frisvad, 2008). Penicillium buchwaldii and P. olsonii generally grow faster than the other species in section Brevicompacta. Furthermore, P. buchwaldii and P. spathulatum have more conspicuously rough and globose to subglobose conidia and have more divergent rami than other species in section Brevicompacta (see illustrations of section Brevicompacta species in Raper & Thom, 1949; Frisvad et al., 1990a; Seifert & Frisvad, 2000; Frisvad & Samson, 2004; Peterson, 2004; Serra & Peterson, 2007 vs. section Ramosa species in Frisvad et al., 1990b, 2006).

The sections Brevicompacta and Ramosa are poorly supported in the ML analysis (< 70%), although Bayesian analysis retrieved a high posterior probability value (1.00 pp). Taxon sampling may play a major role in the phylogenetic inference (Pollock et al., 2002; Zwickl & Hillis, 2002), and perhaps an addition of further species (yet to be discovered) in the cladistic analysis could result in better supported clades. Another option to gain better insight in the relationships is to perform a multigene analysis on these species. Although our phylogenetics analysis shows that P. tularense, P. buchwaldii and P. spathulatum belong in sect. Brevicompactum, a relationship of these species to Penicillium section Ramosa is also evident. P. tularense, P. buchwaldii and P. spathulatum produce similar extrolites and share the ter- to quaterverticillate conidiophores with smooth-walled and globose to subglobose conidia with certain species of sect. Ramosa (Pitt, 1979; Frisvad et al., 1990b; Frisvad & Samson, 2004; Frisvad et al., 2006). On the other hand, some species in section Ramosa produce different combinations of cycloaspeptide, kojic acid and griseofulvin (Frisvad et al., 2006), while these extrolites have not been found in P. buchwaldii, P. spathulatum or P. tularense and any other species in section Brevicompacta.

Most species in section Brevicompacta are very common in soil, but they are also found frequently in foods (Frisvad & Samson, 2004; Table 1). Species in section Ramosa, Turbata and Paradoxa are much more common in soil than in foods (Frisvad et al., 1990b, 2006). Section Penicillium species appears to be originally associated to dung, and some of those may have made a host jump to foods, while species in sections Fasciculata, Roquefortorum, Digitata and Chrysogena are mostly foodborne (Frisvad et al., 2000). Species occurring in indoor environments are concentrated in sections Brevicompacta and Chrysogena (Samson et al., 2010).

The species in section Brevicompacta have asperphenamate in common (except P. fennelliae), but the new species and P. tularense (Table 2) produce a large number of other bioactive extrolites, including communesins, asperentins, benzomalvins, janthitrems, paxillin, quinolactacin and breviones. Several extrolites from the new species and related Penicillia have not been structurally elucidated and thus should be screened for further bioactive drug leads. The production of asperphenamate by many species in Trichocomaceae as major metabolites indicates that some of the fungal producers could be endophytes in the 19 plant species, in which small amounts of asperphenamate have been found, as suggested by Macabeo et al. (2010). The anticancer activity from some medicinal plants may thus be an effect of secondary metabolites from filamentous fungi rather than from metabolites of the plants themselves.


Penicillium buchwaldii

Penicillium buchwaldii Frisvad and Samson sp. nov. Mycobank (MB 800966) (Fig. 3).

Figure 3.

Penicillium buchwaldii. Colonies after 1 week, (a) CYA, (b) MEA, (c) YES, (d–i) conidiophores, (j) conidia.

In: Penicillium subgenus Penicillium section Brevicompacta (Houbraken & Samson, 2011).

Typus: Herb. IMI 304286.

Cultures ex type: CBS 117181 = IBT 6005 = IMI 304286, ex Hordeum vulgare (barley) 16/6 1981, Ørsted, Denmark, J.C. Frisvad.

Etymology: Named in honour of the Danish mycologist N.F. Buchwald.

Diagnostic features: The combination of echinulate thick-walled globose conidia, bi- and ter-ramulate penicilli, pale beige reverse on CYA agar, production of asperphenamate, asperentin, 5′-hydroxyasperentin and communesin A and B.

Similar species: P. tularense, P. brevicompactum and P. bialowiezense.

Description: Colony diameter after 1 week at 25 °C, in mm: CYA: 25–38 (pale beige reverse); CYAS: 25–35; MEA: 25–37; YES: 35–44 (cream yellow with green-olive reverse); CREA: 5–13, very poor growth and no acid production. CYA, 15 °C: 18–30; CYA, 30 °C: microcolonies–10; CYA, 37 °C: no growth. Optimum growth temperature: 21–24 °C. Conidia en masse dark green.

Synnemata or fasciculation: none; Sclerotia: none; Colony texture: velutinous; Conidium colour on CYA: green to dark green; Clear exudate droplets on CYA; Reverse colour on CYA: Cream coloured; Reverse colour on YES: cream yellow; Diffusible colour: none; Ehrlich reaction: weak yellow reaction (no violet reaction); Conidiophores: terverticillate, sometimes biverticillate; Phialides: flask-shaped with short collula, 5.0–8.0 (math formula) × 1.0–2.5 (math formula) μm; Metulae: cylindrical, 4.5–10.0 (math formula) × 1.5–3.0 (math formula) μm; Rami: cylindrical, 11.5–20.0 (math formula) × 2.5–3.5 (math formula) μm; Stipes: smooth to finely rough-walled, 2.5–3.0 (math formula) μm; Conidia: echinulate, thick-walled, globose, 2.0–4.0 (math formula) μm.

Distribution: Denmark, Greenland, Italy, the Netherlands, Senegal, Slovenia, Spain, United Kingdom.

Ecology and habitats: Air in factories, including cake factories, indoor air, air in ceiling, barley, bread, Linum usitatissimum, millet, wheat kernels and wheat flour, margarine container and soil.

Chemotaxonomy (Table 2): All isolates of P. buchwaldii produced asperphenamate. This extrolite has also been reported from 12 species from Trichocomaceae and 19 different plants species (see introduction), and it is also produced by all isolates of P. spathulatum (see below). All isolates of P. buchwaldii also produced Raistrick phenols and asperentin (= cladosporin) and 5′-hydroxyasperentin. The Raistrick phenols have formerly been found in P. brevicompactum and P. bialowiezense, and the asperentins have formerly been reported from Cladosporium cladosporioides (Scott et al., 1971; Jacyno et al., 1993), Aspergillus flavus (Grove, 1972, 1973), Oidiodendron truncatum (John et al., 1999), Microascus tardifaciens (Fujimoto et al., 1999) and Chaetomium globosum (Wang et al., 2006). All isolates of P. buchwaldii also produced communesin B, formerly found in an unidentified Penicillium (Numata et al., 1993), Penicillium expansum (Larsen et al., 1998; Andersen et al., 2004) and P. marinum (Frisvad et al., 2004; originally identified as Penicillium commune, Wigley et al., 2006) and an unidentified fungus (as nomofungin = communesin B) (Ratnayake et al., 2001, 2003). Further communesin derivatives, such as communesin C and D from a Penicillium sp. (Jadulco et al., 2004), communesin D, E and F from P. expansum (Hayashi et al., 2004) and communesin G and H from a Penicillium sp. (Dalsgaard et al., 2005) were not found in P. buchwaldii. Finally, citreoisocoumarin was found in all isolates of P. buchwaldii, except two. Citreoisocoumarin has earlier been found in a Penicillium species (Lai et al., 1991), Penicillium nalgiovense (Larsen & Breinholt, 1999) and Penicillium paneum and Penicillium roqueforti (Frisvad et al., 2004). However, P. buchwaldii does not resemble any of those species listed previously and has its own characteristic profile of extrolites. Furthermore, P. buchwaldii isolates produced some additional extrolites with characteristic UV spectra and retention indices, but these metabolites have not yet been structurally elucidated.

Penicillium spathulatum

Penicillium spathulatum Frisvad and Samson sp. nov. Mycobank MB492650 (Fig. 4).

Figure 4.

Penicillium spathulatum. Colonies after 1 week, (a) CYA, (b) MEA, (c) MEA with 20% sucrose, (d–i) conidiophores, (j) conidia.

In: Penicillium subgenus Penicillium section Brevicompacta (Houbraken & Samson, 2011).

Typus: Herb. CBS 117192.

Cultures ex type: CBS 117192 = IBT 22220, ex French mouldy chestnut (Castanea sp.) (imported to Denmark).

Etymology: Based on the somewhat spathulate rami in the penicilli.

Diagnostic features: Penicillium spathulatum is characterized by higher growth rate at 15 °C than at 25 °C, a higher growth rate in the presence of 5% NaCl (CYAS) as compared to CYA, dark green conidia, an olive grey reverse on CYA, large terverticillate structures with divergent rami and the production of asperphenamate and benzomalvins.

Similar species: P. tularense, P. brevicompactum, P. bialow-iezense and P. buchwaldii.

Description: Colony diameter after 1 week at 25 °C, in mm: CYA: 10–17 mm (grey olivaceous reverse); CYAS: 15–27; MEA: 6–17; YES: 13–22; CREA: 4–9; YES: 16–23 mm (good sporulation, grey green to cream yellow reverse), CREA: 3–10, poor growth and no or weak acid production. CYA, 15 °C: 15–25; CYA, 30 and 37 °C: no growth. Optimum growth temperature: 21 °C.

Synnemata or fasciculation: none; Sclerotia: none; Colony texture: velutinous; Conidium colour on CYA: dark green; Exudate droplets on CYA: none; Reverse colour on CYA: olive grey; Reverse colour on YES: grey green to cream yellow; Diffusible colour: none; Ehrlich reaction: occasionally yellow reaction (no violet reaction); Conidiophores: ter- up to quaterverticillate; Phialides: flask-shaped with short necks, 8.0–10.0 (math formula) × 2.5–3.5 (math formula) μm; Metulae: smooth-walled, cylindrical, 9.5–13.5 (math formula) × 2.5–4.5 (math formula) μm; Rami: typically divergent from the main stipe, 10.0–25.0 (math formula) × 3.0–5.0 (math formula) μm; Stipes: smooth-walled, 2.0–3.5 (math formula) μm; Conidia: (finely) rough-walled, globose to subglobose, 2.5–3.0 (math formula) μm.

Distribution: Easter Island and mainland Chile, Costa Rica, Denmark, Greece, Greenland, India, Japan, the Netherlands, New Zealand, Sweden, USA (Colorado, New Mexico, Wisconsin, Wyoming).

Ecology and habitats: Penicillium spathulatum has been found in beach sand and soil, carrots, dried whale meat, French mouldy chestnut, green mouldy cheese, ice from the Greenland icecap, cheek pouches from kangaroo rats, liver paté, lotus root, margarine container, rain water, salami, sesame seeds, Pleurotus ostreatus, Poa annua, Raphia palm, Tulipa bulb and wheat flour. This species preferably grows on sugar and fat-rich substrates, and we also have encountered it in brines for cheese manufacturing.

Chemotaxonomy: All isolates examined of P. spathulatum produced asperphenamate, and most of the isolates of P. spathulatum produced benzomalvins, formerly isolated from an unidentified Penicillium species (Sun et al., 1994, 1995). Some of the isolates produced breviones, formerly isolated from P. bialowiezense (as P. brevicompactum (Macías et al., 2000a, b) and P. olsonii (Frisvad et al., 2004). Few isolates produced cyclopenin, cyclopeptin and viridicatol, formerly found in several species of Penicillium subgenus Penicillium (Frisvad et al., 2004). Several isolates produced a series of indol alkaloids not seen in any other Penicillium species. Two isolates produced quinolactacin, formerly found in P. citrinum (Takahashi et al., 2000; Houbraken et al., 2010) but also in P. bialowiezense (Frisvad & Samson, 2004). Finally, all isolates produced a series of compounds with UV spectra, indicating perinadine, also reported from P. citrinum (Sasaki et al., 2005). Thus, this species produces extrolites found in both Penicillium subgenus Aspergilloides and subgenus Penicillium as these subgenera are presently understood (Houbraken & Samson, 2011). However, P. spathulatum resembles P. brevicompactum and P. bialowiezense and shares the production of asperphenamate with all but one of the species in section Brevicompacta. It has a unique combination of extrolites. Several P. spathulatum-specific extrolites could only be identified by their UV spectra and retention indices and has yet to be structure elucidated.


We thank The Danish Research Council for Technology and Production Sciences for financial support to Center for Microbial Biotechnology and Ellen Kirstine Lyhne for technical assistance.