Introduction

The use of multigene phylogenies to address complex taxonomic issues is the standard in mycology and currently carries the most weight in the polyphasic species concept used for Penicillium. This is clear from the work of Houbraken and Samson (2011), who reclassified Penicillium into 25 sections based on a four-gene phylogeny. It supersedes the subgeneric classifications of Thom (1930), Raper and Thom (1949), and Pitt (1979), which were based on conidiophore branching patterns. In the DNA barcoding age, which gives scientists in a broad range of disciplines the chance of correct identifications, a sectional classification based on sequence data was an important and invaluable step towards a user-friendly taxonomy for Penicillium. Following this sectional classification, Visagie et al. (2014a) updated the accepted species list for Penicillium and included reference sequences for ex-type strains of the internal transcribed spacer region (ITS), β-tubulin (BenA), calmodulin (CaM), and the DNA-dependent RNA polymerase II second largest subunit (RPB2) gene regions, where available. Also, BenA was proposed as a suitable secondary identification marker to supplement the official DNA barcode ITS (Schoch et al. 2012), which is often not diagnostic for closely related species of Penicillium.

Thom (1930) introduced section Lanata-Divaricata for species with biverticillate conidiophores that usually contain an elongation of the conidiophore’s main axis and metulae that diverge from the axis to form an asymmetrical verticil. As a result, conidiophores can often be interpreted as monoverticillate, although they are in most cases divergently branched biverticilliate conidiophores (also termed divaricate). This group of species is commonly isolated from soil (Thom 1930; Raper and Thom 1949; Pitt 1979; Ramírez 1982; Christensen et al. 2000), but some are frequent on rotting leaf litter (Houbraken et al. 2011). Identifications in the section were traditionally very difficult using morphology. Penicillium janthinellum, the sectional type, and P. simplicissimum, are prime examples. Both were previously characterized by broad species concepts. Pitt (1979) synonymized nine species with P. janthinellum and ten with P. simplicissimum. In another study, Stolk and Samson (1983) synonymized P. janthinellum and P. simplicissimum and linked them and 24 other species to the teleomorph name Eupenicillium javanicum (≡ P. javanicum). However, phylogenetic data showed that E. javanicum sensu lato represents a complex including many distinct species (Peterson 2000; Tuthill et al. 2001; Houbraken et al. 2011; Houbraken and Samson 2011), even though consistent morphological differences are difficult to ascertain. Houbraken et al. (2011) accepted 36 species in the section based on ITS and BenA phylogenies. Unfortunately, Houbraken et al. (2011) did not include phylogenies for calmodulin (CaM) and DNA-dependent RNA polymerase II second largest subunit (RPB2) and we thus considered it advantageous to complete the four-gene data set commonly used in Penicillium for Genealogical Concordance Phylogenetic Species Recognition (GCPSR).

Recently, biodiversity surveys from unique habitats and underexplored countries resulted in description of several new species. One of these unique regions is the fynbos biome situated at the southwestern tip of South Africa. The biome is listed as a UNESCO world heritage site because of its immense biodiversity (Myers et al. 2000), and it boasts more than 9000 plant species, accounting for 44 % of the South African floral inventory (Goldblatt and Manning 2002; Mucina and Rutherford 2006). Over the last couple of years, biodiversity has become an important subject in South Africa, especially in relation to climate change (Midgley et al. 2002; Malcolm et al. 2006). However, knowledge on Penicillium species from South Africa is poor (Schutte 1992), but recent surveys show Penicillium to be a dominant genus in the fynbos region (Allsopp et al. 1987; Visagie et al. 2009, 2013, 2014b; Visagie and Jacobs 2012).

During our survey in the fynbos, 61 Penicillium species were isolated. Six belong to section Lanata-Divaricata, of which four represent previously undescribed species. The aim of this paper is to characterize these using morphology and multigene phylogenies. The new species are compared to their closest relatives and notes provided on their identification. Species names associated with section Lanata-Divaricata are considered based on phylogenies from four genes and suggestions made to modify the accepted species list of Penicillium for this section (Visagie et al. 2014a).

Materials and methods

Sampling and isolations

Penicillium strains were isolated from three fynbos sites that represent unique fynbos types (Musina Mucina and Rutherford 2006). Soil, air, and Protea repens infructescence samples were collected from Stellenbosch (33°56’47”S 18°52’49”), Riverlands Nature Reserve near Malmesbury (33°29’46”S 18°35’16”) and Struisbaai (33°45’06”S 18°58’59”). Isolations were made in similar fashion to methods described by Visagie and Jacobs (2012) and Visagie et al. (2014b).

Reference and ex-type strains were obtained from the public collection of the CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands, the working collection of the Applied and Industrial Mycology department (DTO) of the same institute and from the collection of the United States Department of Agriculture, Agricultural Research Service (USDA-ARS), Peoria, IL, USA(NRRL). Strains used in this study are listed in Table 1.

Table 1 Strains used for phylogenetic analyses of Penicillium section Lanata-Divaricata strains included in the study (n.a. = not available)
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DNA extraction, sequencing, and phylogenetic analysis

DNA was extracted from cultures grown on MEA for 7 days using the ZR Fungal/Bacterial DNA Kit (Zymo Research, CA, USA). PCR amplification of the ITS, BenA, CaM, and RPB2 gene regions and sequencing reactions followed the methods described by Visagie et al. (2014a).

Sequence contigs were assembled and adjusted in CodonCode Aligner v. 4.0.1 (CodonCode Corporation, USA). A sequence database for Penicillium section Lanata-Divaricata species was compiled from newly generated sequences and those previously published in GenBank. Reference sequences of ex-type cultures were obtained from Visagie et al. (2014a). Strains used for phylogenetic comparisons and their GenBank accession numbers are listed in Table 1. Alignments were done using MAFFT v. 7.037b (Katoh and Standley 2013) and aligned data sets analyzed using maximum likelihood (ML) and Bayesian tree inference (BI).

ML analysis was performed in MEGA v. 5.2 (Tamura et al. 2011). The most suitable substitution model for each analysis was chosen using the model test within MEGA, based on the lowest Bayesian information criterion (BIC) value. The initial tree was calculated with the BioNeighbour-Joining (BioNJ) option and subsequent Heuristic search done with the Nearest Neighbour Interchange (NNI) option. Statistical support in nodes was determined using bootstrap analysis with 1000 replicates.

BI analysis was performed in MrBayes v. 3.2.1 (Rondquist and Huelsenbeck 2003), with the most suitable substitution model selected using MrModeltest v. 2.3 (Nylander et al. 2004), based on the lowest Akaike information criterion (AIC) value. The analysis was performed with three sets of four chains. Each analysis was stopped at an average of standard deviation of split frequencies of 0.01. The sample frequency was set at 100, with 25 percent of trees removed as burn-in. ML phylograms were used for representing results with bootstrap (bs) values and posterior probabilities (pp) higher than 80 % bs and/or 0.95 pp represented above branch nodes. Alignments and trees were uploaded onto TreeBase (www.treebase.org) with submission ID 14419.

Morphology

Strains were characterized under standardized growth conditions (Visagie et al. 2014a). Culture media used for characterization include Czapek yeast autolysate agar (CYA), malt extract agar (MEA) (oxoid), yeast extract sucrose (YES) agar, dichloran 18 % glycerol (DG18) agar, CYA supplemented with 5 % NaCl (CYAS), oatmeal agar (OA), and creatine sucrose agar (CREA). Media preparation, inoculation, incubation, and microscope preparations were done as described in Visagie et al. (2014a). Color names and alphanumeric codes used in descriptions refer to the Methuen Handbook of Color (Kornerup and Wanscher 1967). To examine sexual reproductive states, preparations were made from OA.

Microscopic examinations were made using an Olympus BX50 light microscope and Olympus SZX12 stereomicroscope, equipped with an Evolution MP digital microscope camera. Pictures were captured with ImagePro v. 6.0 and microscopic measurements done in Nikon NIS-elements D v. 4.0. In species descriptions, average microscope measurements and their standard deviations are provided between brackets. Photographic plates were prepared in Adobe® Photoshop® CS6. For aesthetic reasons, backgrounds of images were cleaned up using the healing brush tool, without manipulating any of the scientifically relevant areas of images. Colony textures were captured using extended depth of view and processed in Helicon Focus v. 4.2. Line drawings were made using a Nikon Eclipse E800 light microscope with a drawing tube attachment.

Results

Isolations and identifications

Isolations from soil, air and Protea repens infructescence samples collected at three fynbos sites resulted in 1700 Penicillium strains, representing 61 species. Based on their ITS barcodes, six species belong in section Lanata-Divaricata. Two of the species were identified as P. skrjabinii and P. oxalicum. However, four species displayed unique phenotypic characters and grouped separately from known species in the multigene phylogenetic analyses. They are thus described in the taxonomy section as P. annulatum, P. curticaule, P. malacosphaerulum, and P. ortum.

Phylogeny

Fynbos strains were compared with other members of Penicillium section Lanata-Divaricata using four gene regions (Figs. 1 and 2). Aligned data sets for ITS, BenA, CaM, and RPB2 were respectively 501, 442, 488, and 755 bp long when gaps are included. It should be noted that for the newly described P. koreense (You et al. 2014) and P. singorense (Visagie et al. 2014c), only ITS and BenA sequences were included in the analyses. Tree topologies did not differ between ML and BI analyses. For ML, the Kimura 2 parameter with Gamma distributed and Invariant sites (K2 + G + I) was the best fit model for ITS, CaM, and RPB2, whereas the Kimura 2 parameter with Gamma distribution (K2 + G) was the best fit model for BenA. For BI, the General Time Reversible with Gamma distributed and Invariant sites (GTR + G + I) was the best fit model for ITS, CaM, and RPB2 phylogenies, whereas the Symmetrical with Gamma distribution (SYM + G) model was the best fit for the BenA phylogeny.

Fig. 1
figure 1

Maximum likelihood trees based on ITS and BenA, showing the relationship of species in section Lanata-Divaricata. Penicillium glabrum was chosen as outgroup for both phylogenies. Thick branches represent good branch support with Bootstrap values above 80 % and Posterior Probabilities above 0.95 indicated above branches. (T = ex-type; − = support lower than 80 % or 0.95 pp; * = support of 100 % or 1.00 pp). Bold names indicate strains isolated from fynbos. Names in reg indicate strains belonging to new species. Colored boxes indicate the clades to which new species belong (orange = P. janthinellum clade; blue = P. javanicum clade; green = P. rolfsii clade)

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Fig. 2
figure 2

Maximum likelihood trees based on CaM and RPB2, showing the relationship of species in section Lanata-Divaricata. Penicillium glabrum was chosen as outgroup for both phylogenies. Thick branches represent good branch support with bootstrap values above 80 % and posterior probabilities above 0.95 indicated above branches. (T = ex-type; − = support lower than 80 % or 0.95 pp; * = support of 100 % or 1.00 pp). Bold names indicate strains isolated from fynbos. Names in reg indicate strains belonging to new species. Colored boxes indicate the clades to which new species belong (orange = P. janthinellum clade; blue = P. javanicum clade; green = P. rolfsii clade)

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Using the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) concept (Taylor et al. 2000), the results show that Penicillium section Lanata-Divaricata includes 43 species. From Fig. 1 (left), it is clear that several species share identical ITS barcode sequences, in comparison to the other genes, which were able to consistently discriminate 43 species. Clades containing the new species were consistent and well defined in BenA, CaM, and RPB2 phylogenies, and named here as the P. janthinellum (orange block), P. javanicum (blue block) and P. rolfsii (green block) clades. The phylogenies resolved two remaining fynbos strains with P. oxalicum (DTO 182-B1) and P. skrjabinii (DTO 180-I3).

Several species previously thought to belong to this section were also sequenced in this study. The phylogenies showed that P. glaucoroseum, P. cluniae, and P. griseopurpureum are distinct species. The type and ex-type culture of P. glaucoroseum is unavailable, and we propose CBS H-22050 as epitype (ex-epitype CBS 138908 = NRRL 908), considered by Raper and Thom (1949) as representative of the species, in the Names in Current Use (Online Resource 1). In addition, P. asturianum, P. arvense, P. ciegleri, and P. oligosporum are confirmed as synonyms of other species (Frisvad and Filtenborg 1990; Stolk et al. 1990; Houbraken and Samson 2011). No cultures are available for P. es-suveidense, P. populi, P. aragonense, and P. vitale, and we thus follow the synonomies proposed by Pitt (1979) and Stolk et al. (1990), but consider the synonymy of P. aragonense with P. oxalicum, proposed by Stolk et al. (1990), as doubtful because the original description and drawings (Ramírez and Martinez 1981) are not representative of the latter species in our opinion.

Morphology

Fynbos strains produced the typical fast growing colonies of species belonging in Penicillium section Lanata-Divaricata. The strain identified as P. oxalicum (DTO 182-B1) produced large masses of ellipsoidal conidia that easily dislodge in crusts when disturbed. Its phialides are cylindrical and have very short necks. These characters are typical of P. oxalicum. The strain of P. skrjabinii (DTO 180-I3) produced fast growing colonies with variable growth at 37 °C. Its most striking characters are the heavily rough walled conidiophores and conidia. Phialides were observed to be rough walled, which was not previously reported (Pitt 1979; Ramírez 1982); perhaps this reflects the “wild type” of the species, no longer visible in the preserved cultures observed in earlier studies. The four new species were compared to their close relatives in, respectively, the P. janthinellum, P. javanicum and P. rolfsii clades. Colony growth rates and colors produced on different media incubated at different temperatures were taxonomically informative for species identification. With regards to micromorphology, conidial shape and ornamentation were useful characters in the P. janthinellum and P. rolfsii clades, whereas the sexual reproductive states in the P. javanicum clade was most informative. A detailed discussion of these morphological differences is provided in the taxonomy section below.

Species identification

Multigene phylogenies (Figs. 1 and 2) distributed the newly described species among three well-defined clades, named here the P. janthinellum, P. javanicum, and P. rolfsii clades. An identification scheme for all species of these clades is provided here to aid morphological identifications among these close relatives. The scheme includes photoplates of colony characters as well as Tables 2, 3, and 4 summarizing diagnostic characters useful for species identification.

Table 2 Summary of morphological features for distinguishing P. curticaule and P. ortum from its closest relatives
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Table 3 Summary of morphological features for distinguishing P. malacosphaerulum from its close relatives
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Table 4 Summary of morphological features for distinguishing P. annulatum from its close relatives
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The P. janthinellum clade — Two of the new species, P. curticaule and P. ortum, belong to the P. janthinellum clade. The clade includes 12 species with the typical divaricate conidiophores observed for section Lanata-Divaricata as described by Thom (1930). Conidial wall texture and phialide length differed between some species (P. raperi, P. curticaule). However, species identification in this group using micromorphology remains very difficult. Colony morphology and growth rates, especially on CYA at 37 °C, and acid production on CREA, were the most important characters for identification (Table 2, Fig. 3). Furthermore, P. lineolatum and P. ludwigii were found readily to produce a sexual state.

Fig. 3
figure 3

Overview of colony characters in Penicillium section Lanata-Divaricata species in the P. janthinellum clade

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Penicillium janthinellum, P. glaucoroseum, P. ludwigii, P. curticaule, P. raperi, and the newly described P. koreense (You et al. 2014), grow restrictedly on CYA 37 °C (<30 mm). Restricted growth and a brownish to olive reverse on CYA at 25 °C (<30 mm), distinguish P. curticaule and P. raperi from the others. A comparison of these two species reveals that P. raperi grows faster at 37 °C and produces longer phialides than P. curticaule. Penicillium glaucoroseum produces a dark red reverse on CYA and grows faster than P. janthinellum, P. koreense, and P. ludwigii on CYA at 25 and 30 °C. Penicillium ludwigii grows faster on MEA, YES, and CREA than P. janthinellum, and has moderate to strong acid production on CREA. Penicillium koreense shows similar fast growth on CREA to P. ludwigii. However, P. koreense lacks acid production on CREA, does not produce a sexual state and grows more restrictedly than P. ludwigii at 37 °C and on OA.

Penicillium limosum and P. lineolatum readily produce cleistothecia with ascospores, especially on OA. Bright yellow cleistothecia, acid production, and smooth conidia distinguish P. lineolatum from P. limosum. Penicillium brefeldianum has been reported to produce a sexual state (Pitt 1979; Stolk and Samson 1983), but we were unable to induce cleistothecia in this study. However, its colonies grow much faster than both P. lineolatum and P. limosum.

Penicillium cremeogriseum and P. ortum produce acid on CREA, but lacks cleistothecia of P. limosum and P. lineolatum. They have similar conidiophore morphologies and grow strongly on CREA. However, P. ortum consistently grows faster on CYA at 30 and 37 °C, CYAS, YES, and MEA. The remaining species include P. brefeldianum and P. cluniae. Penicillium cluniae displays faster growth on CYA, DG18, and CYAS, making the species readily identifiable, compared to the more restricted growth of P. brefeldianum.

The P. javanicum clade — Penicillium malacosphaerulum belongs to the P. javanicum clade. Colony growth rates and characters of their sexual states were taxonomically most informative (Table 3, Fig. 4). All species in the clade are reported to reproduce sexually, although we did not observe cleistothecia in P. caperatum or P. elleniae. The large dense colonies of P. elleniae on CREA distinguish it from other members in this clade. Also, this species produces smooth walled stipes and spinose conidia, unique for the clade. Penicillium javanicum is the only species in the clade that produces roughened stipes. Ascospores of P. caperatum, P. malacosphaerulum, and P. reticulisporum have two longitudinal flanges, which are absent on P. javanicum ascospores. Penicillium caperatum is reported to produce larger ascospores (5–6 × 3–4 μm) with smooth walls (Stolk and Samson 1983), in comparison to the roughened ascospores of P. javanicum, P. malacospaerulum, and P. reticulisporum. In addition, P. caperatum has moderate acid production on CREA, absent in the other species. Penicillium malacosphaerulum produces smaller ascospores compared to P. reticulisporum. The new species also grows slower on CYA at 25 °C, but faster at 37 °C than P. reticulisporum.

Fig. 4
figure 4

Overview of colony characters in Penicillium section Lanata-Divaricata species in the P. javanicum clade. a–o Overview of sexual reproductive states of P. malacosphaerulum and its close relatives. Included from left to right are cleistothecia, asci and ascospores, a–e. Penicillium javanicum (CBS 341.48). f–j. Penicillium malacosphaerulum (CBS 135121). k–o. Penicillium reticulisporum (CBS 122.68) (— scale bar: in k = 1 mm, applies to a, f, k; in l = 100 μm, applies to b, g, l; in m = 25 μm, applies to c, h, m; in n = 10 μm, applies to d, i, n; in o = 10 μm, applies to e, j, o)

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The P. rolfsii clade — Penicillium annulatum belong to the P. rolfsii clade, which contains species producing terminally biverticillate conidiophores with rough walled stipes. Colony growth rates and conidial shapes are most useful for distinguishing among the species in this clade (Table 4, Fig. 5).

Fig. 5
figure 5

Overview of colony characters in Penicillium section Lanata-Divaricata species in the P. rolfsii clade

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Penicillium annulatum and P. rolfsii grow well on CYA at 37 °C, compared to the generally poor growth observed for other species in the clade. Penicillium rolfsii displays much faster growth on all media and has ellipsoidal conidia, compared to slower growth and globose to subglobose conidia of P. annulatum. Dense white colonies on CREA are characteristic of both P. piscarium and P. svalbardense, but P. piscarium grows faster on CYA, MEA, YES, and OA, and P. svalbardense grows faster on DG18.

The remaining three species, P. ochrochloron, P. pulvillorum, and P. subrubescens, can also be distinguished based on colony growth rates. On CYA, P. ochrochloron grows faster than P. subrubescens. In turn, P. subrubescens grows faster than P. pulvillorum. However, P. ochrochloron grows slower than both species on CYA at 30 °C. In addition, P. ochrochloron and P. rolfsii produce ellipsoidal to fusiform conidia, in contrast with P. pulvillorum’s globose to subglobose conidia. Penicillium pulvillorum generally grows more restrictedly compared to P. subrubescens.

Taxonomy

Penicillium annulatum Visagie & K. Jacobs, sp. nov. Figs. 6 A, 7

Fig. 6
figure 6

Line drawings of the new Penicillium species isolated from the fynbos. A. Penicillium annulatum (CBS 135126). B. Penicillium curticaule (CBS 135127). C. Penicillium malacosphaerulum (CBS 135120). D. Penicillium ortum (CBS 135669). (a = cleistothecia with ascospores; cp = conidiophores and conidia; b = conidiophore branching pattern) (— scale bar: a, cp = 10 μm; b = 50 μm)

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Fig. 7
figure 7

Penicillium annulatum (CBS 135126). a. colonies on CYA, MEA, and YES from left to right (top = obverse, bottom = reverse). b. texture on CYA. c, d. texture on MEA. e–j. conidiophores. k. conidia (— scale bar in e = 50 μm; — scale bar in j = 10 μm, applies to f–k)

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MycoBank: MB809817

ITS barcode: JX091426. The species has unique ITS sequences.

Alternative markers: BenA = JX091514; CaM = JX141545; RPB2 = KF296410.

Etymology: Latin, annulatum meaning surrounded by rings; referring to the rings of sporulation observed in colonies.

Diagnosis — Relatively fast growing colonies on CYA at 25 °C (45–48 mm) and 37 °C (20–30 mm). Stipes rough walled, conidia globose to subglobose and rough walled.

Colony diam, 7 days, in mm — CYA 45–48; CYA 5 °C no germination; CYA 30 °C 48–55; CYA 37 °C 20–30; MEA 40–50; YES 47–52; DG18 24–27; CYAS 25–31; OA 42–45; CREA 28–33.

Macromorphology — CYA, 25 °C, 7days: Colonies low to moderately deep, radially and concentrically sulcate, having a ring-like appearance because of sporulating and non-sporulating areas; margins low, narrow (2 mm), entire; mycelia white; texture floccose; sporulation sparse to moderately dense, conidia dull green (25E4–26E4); exudate dark red, soluble pigment absent, reverse in some isolates dark ruby (12 F8), mostly light to greyish orange (5A5–B5–6). CYA 30 °C, 7 days: Colonies low, lightly radially sulcate, having a ring-like appearance because of sporulating and non-sporulating areas; margin low, narrow (2 mm), entire; mycelia white; texture floccose; sporulation moderately dense, conidia dull to greyish green (25E4–6), sometimes (26E4–6); exudate sometimes present as dark red droplets, soluble pigment absent, reverse dark ruby (12 F8) in central areas for some isolates, others greyish orange (5B4–6) fading into pale yellow (4A3) margin. CYA 37 °C, 7 days: Colonies deep, radially sulcate, concave; margins moderately deep, narrow, irregular; mycelia white; sporulation absent; exudate absent, soluble pigment absent, reverse brownish orange (5C4–6). MEA, 25 °C, 7 days: Colonies 29–38 mm, low to moderately deep, plane; margins low, narrow (2 mm), irregular; mycelia white; texture floccose; sporulation moderately dense to dense, conidia greyish green (25E6–7); exudate absent, soluble pigment absent, reverse greyish yellow (2B3–4). YES, 25 °C, 7 days: Colonies low, lightly radially sulcate, having a ring-like appearance because of sporulating and non-sporulating areas, but less obvious than those on CYA; margins low, narrow (2 mm), entire; mycelia white; texture floccose; sporulation sparse to moderate, conidia greyish green (25B4–5–30D5); exudate absent, soluble pigment absent, reverse dull yellow to yellowish white (3A3–B3–B4). CREA 25 °C, 7 days: Colonies not producing acid.

Micromorphology — Conidiophores bi- and terverticillate, often with subterminal branches produced; stipes rough walled, 180–750 × 3–4.5 μm; rami/branches divergent, 6–40 × 3–4.5 μm (19.8 ± 6.73); metulae 3–6 per stipe/branch, appressed to divergent, angle 28–80° (52 ± 11.3°), great variation in length in the same conidiophore, 8–20 × 2.5–4.5 μm (11.5 ± 1.89 × 3.5 ± 0.43), vesicle 3–4.5 μm (3.38 ± 0.41); phialides ampulliform, 4–5 per metula, 6–8 × 2–3.5 μm (7.1 ± 0.5 × 2.8 ± 0.25); conidia rough walled, globose to some subglobose, 2.5–3 × 2–3 μm (2.5 ± 0.12 × 2.4 ± 0.13), average width/length ± stdev = 0.93 ± 0.04, n = 116.

Holotypus — South Africa, Stellenbosch, (33°56’47”S 18°52’49”), from air sample, 14 March 2009, collected and isolated by CM Visagie, CBS H-21333 (ex-typus: CBS 135126 = CV 37 = DTO 180-G7).

Additional cultures examined — South Africa, Stellenbosch, (33°56’47”S 18°52’49”), from mite in Protea repens infructescence, 14 March 2009, collected and isolated by CM Visagie, CBS 135125 = CV 187 = DTO 181-C3; South Africa, Stellenbosch, (33°56’47”S 18°52’49”), from soil, 14 March 2009, collected and isolated by CM Visagie, CBS 135123 = CV 548 = DTO 181-I1; South Africa, Struisbaai, (33°45’06”S 18°58’59”), from soil, 14 August 2009, collected and isolated by CM Visagie, CBS 135124 = CV 1707 = DTO 183-E9.

Penicillium curticaule Visagie & K. Jacobs, sp. nov. Figs. 6B & 8

Fig. 8
figure 8

Penicillium curticaule (CBS 135127). a. colonies on CYA, MEA, and YES from left to right (top = obverse, bottom = reverse). b, c. texture on MEA. d–j. conidiophores. k. conidia (— scale bar in d = 10 μm; — scale bar in j = 10 μm, applies to e–k)

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MycoBank: MB809818

ITS barcode: FJ231021. The species has unique ITS sequences.

Alternative markers: BenA = JX091526; CaM = JX141536; RPB2 = KF296417.

Etymology: Latin, curticaule: Curtus meaning short, caulis meaning stemmed; referring to the short stipes produced by the species.

Diagnosis — Relatively slow growing colonies on CYA at 25 °C (23–26 mm) and 37 °C (3–8 mm). Colonies on OA have olive yellow to olive to olive brown color at margins. Conidiophores with very short stipes (9–20 μm) and phialides (5–7.5 μm).

Colony diam, 7 days, in mm — CYA 23–26; CYA 5 °C germination; CYA 30 °C 30–35; CYA 37 °C 3–8; MEA 18–21; YES 30–35; DG18 16–18; CYAS 10–11; OA 20–22; CREA 20–25.

Macromorphology — CYA, 25 °C, 7 d: Colonies low to moderately deep, radially sulcate, random furrows also present; margins low, narrow (2 mm), yellowish olive; mycelia white; texture floccose; sporulation sparse to moderate, conidia dull green (25D3–4); exudate absent, soluble pigment absent, reverse olive yellow to olive to olive brown (3D7–8–4D7–8). CYA, 30 °C, 7 days: Colonies low to moderately deep, irregular furrows present; margins low, narrow (1–2 mm), having a yellowish olive color, entire; mycelia white; texture floccose; sporulation sparse, conidia greyish turquoise (25C3); exudate absent, soluble pigment absent, reverse Brown (6E7–8) at centre, olive brown (4D8) and greenish yellow (1A7) elsewhere. CYA, 37 °C, 7 days: Microcolonies of white mycelia present. MEA, 25 °C, 7 days: Colonies low, plane, slightly raised at centre; margins low, narrow (2–3 mm); mycelia white; texture floccose, loosely funiculose; sporulation moderately dense, conidia greyish to Dull Green (25C3–D4–5); exudate absent, soluble pigment absent, reverse olive to Yellowish Brown (4E4–5E4) at centre, margin greyish yellow (3B6). YES, 25 °C, 7d: Colonies low, radially sulcate, random furrows also present; margins low, narrow (1–2 mm), entire, yellowish olive color; mycelia white; texture floccose; sporulation sparse to moderate, conidia dull green (25D3–4); exudate absent, soluble pigment absent, reverse olive yellow to olive to olive brown (3D7–8–4D7–8). CREA, 25 °C, 7 days: Colonies not producing acid.

Micromorphology — Conidiophores mostly very short and monoverticillate, although these might be considered side branches of divaricate type conidiophores, very few true biverticillate conidiophores were observed; coiling of mycelia often observed; stipes/metulae smooth walled, 9–20 × 2–3.5 μm (13.3 ± 3.5 × 2.6 ± 0.33), vesicle 2.5–3.5 μm (2.8 ± 0.28), biverticillate conidiophore stipes 50–200 μm, only 2 metulae present, often a solitary phialide borne on same level as metula; phialides 1–5 per stipe/metula, ampulliform, 5–7.5 × 2–3 μm (6.4 ± 0.78 × 2.8 ± 0.28); Conidia rough walled, broadly ellipsoidal, 2–3 × 2–2.5 μm (2.6 ± 0.17 × 2.2 ± 0.14), average width/length = 0.86 ± 0.05, n = 77.

Holotypus — South Africa, Malmesbury, (33°29’46”S 18°35’16”), from soil, 21 February 2007, collected and isolated by CM Visagie, CBS H-21334 (ex-typus: CBS 135127 = CV 2842 = DTO 180-D3 = DAOM 241159).

Additional cultures examined — South Africa, Malmesbury, (33°29’46”S 18°35’16”), from soil, 21 February 2007, collected and isolated by CM Visagie, CBS 135128 = CV 2857 = DTO 184-D1, CBS 135129 = CV 2858 = DTO 180-F2.

Penicillium glaucoroseum Demelius, Verh. Zool.-Bot. Ges. Wien 72: 72, 1923 (1922)

MycoBank: MB158423

ITS barcode: KF296407. The species share identical ITS sequences with P. ortum and P. cremeogriseum.

Alternative markers: BenA = KF296469; CaM = KF296400; RPB2 = KF296430.

Lectotypus — Fig. 3. (Demelius, Verh. Zool.-Bot. Ges. Wien 72: 73, 1923 (1922)), hic designatus.

Epitypus — USA, Virginia, from soil, unknown date and collector, CBS H-22050 (ex-epitypus: CBS 138908 = NRRL 908), hic designatus.

Penicillium malacosphaerulum Visagie & K. Jacobs, sp. nov. Figs. 6C & 9

Fig. 9
figure 9

Penicillium malacosphaerulum (CBS 135121). a. colonies on CYA, MEA, and YES from left to right (top = obverse, bottom = reverse). b, c. texture on MEA. d. cleistothecia. e–i. conidiophores. j. conidia. k. asci and ascospores (— scale bar in d = 50 μm; — scale bar in i = 10 μm, applies to e–i; sScale bar in k = 10 μm)

Full size image

MycoBank: MB809819

ITS barcode: FJ231026. The species has unique ITS sequences, but they are very similar to ITS barcodes of P. caperatum and P. reticulisporum.

Alternative markers: BenA = JX091524; CaM = JX141542; RPB2 = KF296438.

Etymology: Latin, malacosphaerulum: malacos meaning soft, sphaerula meaning small ball; referring to the soft yellow ascocarps produced by this species.

Diagnosis — Relatively slow growth on CYA at 25 °C (28–36 mm), but fast growth at 30 °C (40–45 mm) and 37 °C (30–35 mm). Colonies produce yellow soluble pigment. Cleistothecia dark yellow to brown, ascospores finely roughened with two longitudinal flanges present and relatively small (2.5–3.5 × 2–3 μm).

Colony diam, 7 days, in mm — CYA 28–36; CYA 5 °C sometimes germination; CYA 30 °C 40–45; CYA 37 °C 30–35; MEA 26–32; YES 33–43; DG18 12–15; CYAS 19–22; OA 25–27; CREA 25–30.

Macromorphology — CYA, 25 °C, 7 days: Colonies low to moderately deep, radially, and concentrically sulcate; margins low, wide (3 mm), entire; mycelia white and inconspicuously yellow; texture floccose; sclerotia produced especially near colony centre, giving colony a greyish yellow (4C4) color, after 2–4 weeks developing into cleistothecia, sporulation absent; exudate dark brown (8 F8), soluble pigment bright yellow, reverse yellowish brown (5D7–E7) at centre, fading into greyish yellow (4B5) into a yellow (2A7–8) margin. CYA, 30 °C, 7 days: Colonies similar to that of CYA at 25 °C. CYA, 37 °C, 7 days: Colonies low to moderately deep, radially, and concentrically sulcate; margins low, narrow (1 mm), somewhat irregular; mycelia white; texture floccose; sclerotia abundantly produced, giving a greyish yellow (4C4) color to colonies; sporulation moderate, conidia dull green (26D3); exudate a few clear droplets produced, soluble pigment bright yellow, greyish green (1C5) at centre, elsewhere greenish to greyish yellow (1A7–B7), in some isolates brownish orange (5C5) at centre. MEA, 25 °C, 7 days: Colonies low, plane; margins low, narrow (1–2 mm), irregular; mycelia white, inconspicuously yellow; texture floccose; sclerotia abundantly produced, which develop into mature cleistothecia after 2–4 weeks, color ranging from light yellow to greyish orange (2A4–5B3); sporulation absent, conidiophores developing only after 14 days of incubation, conidia indeterminable; exudate absent, soluble pigment absent, reverse yellow (3A8) near centre fading into yellowish white (3A2) margin. YES, 25 °C, 7 days: Colonies low, radially and concentrically sulcate, randomly furrowed as well, sunken in at centre; margins low, narrow (1–2 mm), entire; mycelia white, inconspicuously yellow; texture floccose; sclerotia abundantly produced, giving a greyish yellow (4C4) color; sporulation absent to sparse, conidia indeterminable; exudate absent, soluble pigment yellow, reverse mostly light yellow (2A5–3A5) with yellow (3A6–7) areas present. CREA, 25 °C, 7 days: Colonies not producing acid.

Micromorphology — Conidiophores irregularly biverticillate and divaricate; stipes smooth walled, 100–500 × 2–3 μm, sometimes shorter, then 30–80 μm; metulae mostly 2 with occasionally 3 per stipe, verticils appressed to divergent, angle 22–41° (32 ± 6.7°), 6.5–22 × 2–3.5 μm (14.1 ± 4.4 × 2.6 ± 0.33), vesicle 2.5–4 μm (3.3 ± 0.34); phialides ampulliform, 3–5 per metula/stipe, (6)7–10 × 2–3(3.5) μm (7.8 ± 0.84 × 2.77 ± 0.27); conidia smooth walled, globose to subglobose, 2.5–3 × 2.5–3 μm (2.6 ± 0.14 × 2.4 ± 0.16), average width/length = 0.92 ± 0.04, n = 81; cleistothecia abundantly produced on most media, brownish to dark yellowish, 50–140 × 50–130 μm (83 ± 19.61 × 73 ± 17.6); asci borne singly, 5.5–10 × 4.5–7 μm; ascospores finely rough walled, with two longitudinal flanges, subglobose to broadly ellipsoidal, 2.5–3.5 × 2–3 μm (3 ± 0.18 × 2.5 ± 0.17), average width/length = 0.82 ± 0.04, n = 83.

Holotypus — South Africa, Malmesbury, (33°29’46”S 18°35’16”), from soil, 21 February 2007, collected and isolated by CM Visagie, CBS H-21332 (ex-typus: CBS 135121 = CV 2855 = DTO 180-E6 = DAOM 241161).

Additional cultures examined — South Africa, Malmesbury, (33°29’46”S 18°35’16”), from soil, 21 February 2007, collected and isolated by CM Visagie, CBS 135120 = CV 2836 = DTO 180-D1, CBS 135122 = CV 2848 = DTO 180-E1.

Penicillium ortum Visagie & K. Jacobs, sp. nov. Figs. 6D & 10

Fig. 10
figure 10

Penicillium ortum (CBS 135669). a. colonies on CYA, MEA, and YES from left to right (top = obverse, bottom = reverse). b, c. texture on MEA. d–i. conidiophores. j. conidia (— scale bar in d = 50 μm; — scale bar in i = 10 μm, applies to e–j)

Full size image

MycoBank: MB809820.

ITS barcode: JX091427. This species shares identical barcodes with P. cremeogriseum, and has very similar barcodes to P. glaucoroseum, P. ludwigii, P. janthinellum, and P. cluniae.

Alternative markers: BenA = JX091520; CaM = JX141551; RPB2 = KF296443.

Etymology: Latin, ortum meaning sunrise; referring to the colony appearance on YES resembling that of a “sunny side up egg”.

Diagnosis — Relatively fast growth on CYA at 30 °C (36–50 mm) and 37 °C (35–40 mm). Strong growth on CREA with moderate acid production. Reverse on CYA olive to brownish orange. Conidia smooth walled.

Colony diam, 7 days, in mm — CYA 28–40; CYA 5 °C germination; CYA 30 °C 36–50; CYA 37 °C 35–40; MEA 48–53; YES 40–50; DG18 20–27; CYAS 30–36; OA 40–45; CREA 25–30.

Macromorphology — CYA, 25 °C, 7 days: Colonies moderately deep, lightly radially sulcate; margins low, narrow (1–2 mm), entire; mycelia white at margin, light yellow to yellowish grey elsewhere; texture floccose; sporulation absent to sparse, conidia dull green (26D3); exudate mostly absent, but sometimes clear, soluble pigment yellow, reverse olive (3E8) becoming lemon yellow (3B8) near margins, brownish orange (7C6) areas also present. CYA 30 °C, 7 days: Colonies moderately deep, radially sulcate, having a yellowish colour; margins low, narrow 2–3 mm, entire; mycelia white; sporulation absent; exudate yellow, soluble pigment absent, reverse brown (5 F7–8) at colony centre, elsewhere olive to dark yellow (3C6–8–4C6–8), with yellow (2A6) margin. CYA 37 °C, 7 days: Colonies moderately deep, radially and concentrically sulcate, having a pale yellow (1A3) colour; margins low, narrow (1–2 mm), entire; mycelia white; texture floccose; sporulation sparse, only near colony margins, conidia greenish grey (29C2–30B2); exudate yellow, soluble pigment yellow, reverse olive brown (4E8) at centre, fading into olive to greyish yellow (3C6–4C6). MEA, 25 °C, 7 days: Colonies moderately deep, plane; margins low, narrow, entire; mycelia white at margin, light yellow elsewhere; texture floccose; sporulation sparse to moderate, conidia dull green (26D3); exudate absent, soluble pigment absent, reverse olive yellow (3C8) becoming yellow (3A7) near margins. YES, 25 °C, 7 days: Colonies moderately deep, radially sulcate, also randomly furrowed; margins low, very narrow (1–2 mm), entire; mycelia white at margin, light yellow elsewhere; texture floccose; sporulation sparse to sometimes absent, conidia dull green (26D3); exudate absent, soluble pigment absent, reverse areas of brown to dark brown (5 F8–6 F8), elsewhere varying from greyish yellow to orange yellow (4B6–8), margins light yellow (4A4). CREA 25 °C, 7 days: Colonies producing moderate acid.

Micromorphology — Conidiophores biverticillate with a large number of subterminal side branches formed that may be mono- or biverticillate; stipes smooth walled, 150–550 × 2–3 μm, side branched “conidiophore” stipes 18–90 μm; metulae 2 to 4 per stipe, mostly divergent, angle 19–50° (34 ± 9.2°), 10–30 × 2–3 μm (18.1 ± 4.91 × 2.7 ± 0.27), vesicle 2.5–4 μm (3.1 ± 0.33); phialides ampulliform, 3 to 7 per metula, 6.5–9(10) × 2–3 μm (7.6 ± 0.75 × 2.8 ± 0.21); conidia smooth walled, globose to subglobose, 2.5–3.5 × 2.5–3.5 μm (2.9 ± 0.2 × 2.7 ± 0.2), average width/length = 0.92 ± 0.04, n = 76.

Holotypus — South Africa, Stellenbosch, (33°56’47”S 18°52’49”), from soil sample, 14 March 2009, collected and isolated by CM Visagie, CBS H-21602 (ex-typus: CBS 135669 = CV 102 = DTO 180-I9).

Additional cultures examined — South Africa, Stellenbosch, (33°56’47”S 18°52’49”), from soil sample, 14 March 2009, collected and isolated by CM Visagie, CBS 135668 = CV 71 = DTO 180-H7, CBS 135667 = CV 95 = DTO 180-I7; South Africa, Stellenbosch, (33°56’47”S 18°52’49”), from Protea repens infructescence bract, 14 March 2009, collected and isolated by CM Visagie, CBS 135670 = CV 391 = DTO 181-F5.

Discussion

Traditionally, Penicillium section Lanata-Divaricata is taxonomically challenging because of morphological similarities between species and variation within a species. This resulted in Pitt (1979) synonomising a large number of species with P. janthinellum and P. simplicissimum citing the roughened stipes and more regular branching pattern of the latter diagnostic in the latter in comparison with the smooth walled stipes and divaricate branching of P. janthinellum. Stolk and Samson (1983) on the other hand considered both P. janthinellum and P. simplicissimum as the anamorph state of Eupenicillium javanicum and thus lumped 24 species, irrespective of conidiophore morphologies. Even though these authors attempted to simplify identifications in this difficult group, it always remained problematic. One problem associated with the group is the variation observed from strain to strain, wether it be slightly different colors in colonies or growth rates. This creates the difficulty of not knowing wether strains with morphological variation represented a previously described species or not. Only with the incorporation of DNA sequence data into the species concept used for Penicillium were we able to satisfactorily resolve these problems and make identifications easier. This is because of the clearer species boundaries inferred from phylogenies that makes morphological comparisons easier.

The study presented here compared section Lanata-Divaricata species based on phylogenies from four genes (ITS, BenA, CaM, and RPB2) and morphology, applying GCPSR to the phylogenetic data. The phylogenies presented here supported the acceptance of 36 species by Houbraken et al. (2011), and demonstrated distinctiveness of P. cluniae, P. glaucoroseum, and P. griseopurpureum. We introduced four new species isolated from the fynbos biome in South Africa as P. annulatum, P. curticaule, P. malacosphaerulum, and P. ortum, which are phylogenetically resolved in consistent clades with their respective close relatives. These clades were designated here as the P. janthinellum, P. javanicum, and P. rolfsii clades. All species from these clades were characterized to more precisely define them morphologically and aid future identifications. Generally, colony growth rates on different media incubated at different temperatures were informative characters. This is especially true for growth on CYA at 37 °C. Also, acid production and colony characters on CREA were valuable for distinguishing among closely related species. For the P. javanicum clade, characters of the sexual reproductive structures distinguish among species, whereas conidiophore morphologies were informative in the P. rolfsii clade. This includes the size of asci and ascospores, as well as ascospore ornamentation, similar to the conclusions of Udagawa and Horie (1973). Generally, conidiophore morphology was very difficult to consistently interpret; mainly because of conidiophore branching that was found to be so diverse and variable within a species that it becomes uninformative for species identification. This was evident in especially the P. janthinellum and P. javanicum clades. In the P. janthinellum clade, only phialide length and conidial wall texture was informative. Pitt (1979) emphasized the long slender phialide necks and divaricate branching in P. janthinellum. However, the long phialides were also observed in P. lineolatum and P. ludwigii and while divaricate branching is characteristic of all species in this clade, it is not restricted to it. In the P. javanicum clade only stipe and conidial wall texture was informative.

Dodge (1933) described P. brefeldianum based on CBS 235.81. This particular strain lost the ability to reproduce sexually, and Pitt (1979) neotypified the species with CBS 233.81. Houbraken and Samson (2011) showed that CBS 233.81 is phylogenetically identical to P. caperatum (CBS 443.75) and reinstated CBS 235.81 as type for P. brefeldianum. In our study, none of these strains were able to reproduce sexually. Phylogenetically, P. brefeldianum and P. caperatum are respectively resolved in the P. janthinellum and P. javanicum clades, even though morphologically they are very similar. The main difference is that P. caperatum typically produces acid on CREA and grows slightly slower on MEA than P. brefeldianum.

Previous phylogenetic studies focused on section Lanata-Divaricata did not include P. glaucoroseum, P. cluniae, or P. griseopurpureum (Houbraken et al. 2011; Houbraken and Samson 2011). Houbraken and Samson (2011), however, suggested that unpublished data showed that they are distinct species that are appropriately classified in the section. Here we confirm this, with P. glaucoroseum and P. cluniae resolved in the P. janthinellum clade and P. griseopurpureum closely related to P. daleae. Demelius (1922) described P. glaucoroseum and emphasized the production of rosy crystals and granules in colonies. Based on the original description, Thom (1930) placed the species in his P. janthinellum series and considered strain NRRL 908 a good representative for P. glaucoroseum. Unfortunately, none of Demelius’ authentic material is obtainable. In common with Thom (1930) and Raper and Thom (1949), we consider NRRL 908 to be in good condition, with the culture producing the dark reddish to purple reverse and irregular conidiophores as described by Demelius (1922). As such, we lectotypified P. glaucoroseum based on Demelius’ Fig. 3 (1922) and epitypified it with the dried specimen CBS H-22050 (ex-epitype NRRL 908 = CBS 138908) above.

Ramírez and Martinez (1981) described Penicillium aragonense, with the ex-type strain submitted and preserved in the CBS-KNAW culture collection under accession number CBS 171.81. However, this culture is badly contaminated with P. glabrum. Based on the original description, Stolk et al. (1990) synonymized the species with P. oxalicum. In contrast, our opinion is that the drawing and original description of P. aragonense (Ramírez and Martinez 1981) do not match P. oxalicum. Even though the large, ellipsoidal conidia (4–6 × 3.2–5 μm) seem similar to those of P. oxalicum, the description does not resemble the conidiophore shapes or the large masses of conidia in colonies typical of P. oxalicum. Based on this and the absence of additional material, we have to consider its identity uncertain. In the same paper, Ramírez and Martinez (1981) described P. asturianum. Although their description and drawings resemble P. oxalicum, they considered it a close relative of P. janthinellum. Subsequently, Stolk et al. (1990) reduced P. asturianum to synonymy with P. oxalicum. Morphological characters combined with the multigene phylogeny presented here confirm this species as a synonym of P. oxalicum.

This study provided phylogenies to species belonging in Penicillium section Lanata-Divaricata and reported on four new species. The paper follows previous publications reporting new species from the diverse fynbos biome situated in South Africa (Visagie et al. 2013, 2014b; Houbraken et al. 2014), and represents a small proportion of undescribed Penicillium species isolated during a survey of the genus in the fynbos, which will be described in future publications.