Three new additions to the genus Talaromyces isolated from Atlantis sandveld fynbos soils.

During a survey of Penicillium spp. in soils from the diverse fynbos region in the Western Cape, South Africa, a number of previously undescribed species were isolated. Three of these belong to subg. Biverticillium sensu Pitt, recently incorporated into its previously associated teleomorph genus, Talaromyces s.str. These species displayed symmetrical biverticillate penicilli, acerose phialides and poor growth at reduced water activity, typical of this group. Morphological characters of the new species were compared to known Talaromyces species. The ITS and β-tubulin gene regions were used for phylogenetic comparisons, which confirmed the distinct nature of the three fynbos soil species described here as Talaromyces chloroloma sp. nov., T. ptychoconidium sp. nov. and T. solicola sp. nov., respectively. Talaromyces chloroloma is typically recognised by its strongly funiculose colony texture and after prolonged incubation, synnemata can be observed on CYA. Talaromyces ptychoconidium is characterised by closely appressed conidiophores that produce spirally rough-walled conidia, while T. solicola typically struggle to grow on CYA and is distinguished from similar species by its prominently rough-walled, spheroid conidia.

INTRODUCTION

The fynbos biome situated in the Western Cape, South Africa, is considered one of the most botanically diverse habitats on earth with approximately 9 030 vascular plant species. This accounts for 44 % of the total floral inventory of South Africa (Goldblatt & Manning 2002). Based on the association between plants and fungi, Hawksworth (1991, 2001) estimated the scope of fungal diversity on earth to be 1.5 million species, using a plant vs fungus ratio of 1 : 6 (Hawksworth 1991, 2001). Crous et al. (2006) suggested this ratio to be 1 : 7 for South Africa and estimated that 171 500 fungal species may be present in these unique habitats. Extrapolating this ratio to the vascular plants from the fynbos area, approximately 63 210 fungal species are expected to occur in this botanically diverse region of the Western Cape. However, only a fraction of this estimate has been documented from this habitat thus far.

Similar to other global soil surveys (Christensen et al. 2000), Penicillium was found to be one of the dominant fungal genera in fynbos soil, contributing to the majority of the species diversity in this niche. Previous surveys conducted in the fynbos, focused on genera other than Penicillium s.l., with Allsopp et al. (1987) representing the only study identifying strains to species level. Considering the inherent diverse and unique nature of the fynbos biome one would expect that the soils harbour a large number of fungi, particularly species that are unknown to science (Hawksworth 1991, 2001, Crous et al. 2006).

Penicillium subg. Biverticillium is one of the better-defined subgenera of Penicillium s.l., with 23 species accepted by Pitt (1979). These species are easily distinguished from other groups by their distinct symmetrical biverticillate conidiophores that bear typical acerose phialides, while some species produce ampulliform-like phialides that end in a long and fine neck. Species characteristically have a metula to phialide length ratio of ± 1–1.2 and poor growth at reduced water activity (Pitt 1979, Pitt & Hocking 1997, Samson et al. 2011). Penicillus dimensions and shapes within species of this group are very similar. Colony characters were, therefore, often the criterion used to distinguish between closely related species, since they often contain brightly pigmented features such as mycelia, soluble pigments, exudates and/or reverse colony colour (Pitt 1979, Pitt & Hocking 1997). Penicillium s.l. has been widely accepted as being polyphyletic, with two distinct phylogenetic clades that correspond to the teleomorphic genera Eupenicillium and Talaromyces, respectively (LoBuglio et al. 1993, Berbee et al. 1995, Peterson 2000, Heredia et al. 2001, Seifert et al. 2004, Visagie et al. 2009, Houbraken & Samson 2011, Samson et al. 2011). In accordance with changes made to the ICBN and movement towards single-name nomenclature for fungi (Hawksworth et al. 2011, Norvell 2011), Houbraken & Samson (2011) incorporated Eupenicillium as a synonym of Penicillium s.str. and transferred species in Penicillium subg. Biverticillium to Talaromyces s.str. as new combinations (Samson et al. 2011), accepting 74 species and excluding species, i.e. P. rubrum and P. lignorum, with questionable taxonomic positions (Samson et al. 2011).

During a survey of the microbial diversity of fynbos soils, eight dis- tinct taxa belonging to Talaromyces were isolated from Atlantis sandveld fynbos soil. Three of the isolated species were found to be novel. The aim of this study was, therefore, to compare and describe the new species with previously described Talaromyces spp. using morphological and phylogenetic characters. In addition to this, an identification key to the new species and their close relatives is included.

MATERIALS AND METHODS

Isolations

Strains were collected from sandy fynbos soil, situated near Malmesbury in the Western Cape, at Camphill Village (S 33,59787°; E 18,56433°), Kalbaskraal (S 33,57061°; E 18,62861°), Pella (S 33,51022°; E 18,55236°) and Riverlands (S 33,49066°; E 18,58388°). At each sampling site, random soil samples were collected from different plots. Five grams of each soil sample were added to 100 mL dH2O. A dilution series were prepared from this, with the 10-1 and 10-2 dilutions plated onto Potato-Dextrose Agar (PDA; Biolab, Johannesburg, South Africa), containing 50 ppm Streptomycin (Applichem, South Africa) and 100 ppm Chloramphenicol (Applichem, South Africa). Plates were incubated at 25 °C for 6–7 d, after which colonies resembling those of Penicillium were transferred to Oatmeal Agar (OA). Strains were incubated for a further 7 d at 25 °C, after which single spore cultures were prepared.

Morphology

Spore suspensions of strains were prepared in a semi-solid agar (0.2 % agar; 0.05 % Tween 80). Inoculations, from these spore suspensions, were done in three point style on Czapek Yeast Autolysate Agar (CYA), Malt Extract Agar (MEA: after Blakeslee 1915), Yeast Extract Sucrose Agar (YES) and 25 % Glycerol Nitrate Agar (G25N) (Pitt 1973, 1979, Samson & Pitt 1985, Frisvad & Samson 2004). Media were modified by adding copper-sulphate (CuSO4·5H2O) and zink-sulphate (ZnSO4·7H2O) (Frisvad & Samson 2004). The inoculated Petri dishes (90 mm), containing 20 mL of media, were incubated at 25 °C (CYA, MEA, YES, G25N), 5 °C (CYA) and 37 °C (CYA) (Pitt 1979, Samson & Pitt 1985), in the dark with plates left unwrapped to allow for sufficient aeration (Okuda et al. 2000). Additional inoculated CYA and MEA plates were incubated in incidental light at room temperature (± 23 °C) for 7–21 d. Strains were characterised and described following the methods of Pitt (1979), Samson & Pitt (1985), Okuda et al. (2000) and Frisvad & Samson (2004). Colour names and codes follow that of Kornerup & Wanscher (1967). Reference strains for T. calidicanius (CBS 112002), T. ceci- dicola (KAS 509), T. dendriticus (KAS 849 & KAS 1190), T. diversus (CBS 320.48), T. duclauxii (MUCL 28672), T. erythromellis (CBS 644.80), P. lignorum (CBS 709.68), T. palmae (CBS 442.88), T. phialosporus (CBS 233.60), T. panamensis (CBS 128.89), T. pseudostromaticus (CBS 470.70), T. ramulosus (CMV 113 = DAOM 241015, CV 2804, CV 2805), T. rugulosus (CBS 255.31) and T. purpureus (CBS 475.71), were examined and com- pared to the new species.

Phylogenetic analysis

DNA was extracted from strains grown on MEA for 7 d using the ZR Fungal/Bacterial DNA Kit (Zymo Research, California, USA). The subsequent PCR of the ITS1–5.8S–ITS2 rDNA region were prepared in 25 μL total volume reactions and consisted of 2.5 μL 10X Kapa Taq High Yield Buffer A, 2.5 U Kapa Taq (Kapa Biosystems, Woburn, USA), 250 μM dNTPs and 0.25 μM of primers ITS1 and ITS4, respectively (White et al. 1990). The thermal cycle profile had an initial denaturing step at 94 °C for 5 min, followed by 36 cycles at 94 °C for 45 s, 56 °C for 45 s, 72 °C for 60 s, followed by a final elongation step at 72 °C for 10 min. The β-tubulin gene was amplified using the same reagents as for the ITS PCR, except for primers Bt2a and Bt2b used (Glass & Donaldson 1995). The thermal cycle profile had an initial denaturing step at 94 °C for 3 min, followed by 36 cycles at 94 °C for 45 s, 52 °C for 45 s, 72 °C for 60 s, and was completed with a final elongation step at 72 °C for 10 min.

PCR products were purified using the MSB®Spin PCRapace (Invitek, Berlin) kit. Sequencing reactions of the PCR products were set up using a Big Dye terminator cycle sequencing premix kit (Applied Biosystems, CA). The thermal cycle profile had an initial denaturing step at 94 °C for 5 min, followed by 25 cycles at 94 °C for 10 s, 55 °C for 10 s and 60 °C for 4 min. Sequence reactions were analysed with an ABI PRISM 310 genetic analyser, with the subsequent sequence contigs assembled in CodonCode Aligner (v. 3.7.1.1, Codon Code Corporation). The ITS and β-tubulin sequences of the fynbos soil strains were included in datasets containing sequences from GenBank of known Penicillium subg. Biverticillium and Talaromyces species (LoBuglio et al. 1993, Peterson 2000, Heredia et al. 2001, Seifert et al. 2004, Visagie et al. 2009).

Datasets were aligned in MAFFT v. 6.850b (Katoh et al. 2009) with the L-INS-i option selected. All of the aligned datasets can be obtained from TreeBASE (Submission ID 11467). Sequence analysis was done in PAUP v. 4.0b10 (Swofford 2000), using the BioNJ option for calculating a single tree for each dataset (Gascuel 1997). Confidence in nodes was calculated using a bootstrap analysis of 1 000 replicates. Talaromyces thermophilus was chosen as outgroup for the ITS phylogeny (Heredia et al. 2001). For β-tubulin, clade specific phylogenies were done which gave a better alignment than a total phylogeny. Penicillium lignorum and T. phialosporus were chosen as outgroups for the T. solicola clade phylogeny. For the T. chloroloma / T. ptychoconidium clade, synnemata-producing species T. calidi- canius, T. duclauxii, T. palmae and T. panamensis were chosen as outgroups.

RESULTS

Isolations made from soil dilutions yielded 434 Penicillium s.l. strains. The isolates were placed into their respective taxa, using colony characters on CYA, MEA and YES. Sixty-six strains represented seven distinct morphological taxa belonging to Talaromyces. All of them had biverticillate conidiophores with acerose phialides and showed poor growth at reduced water activity. Taxa were identified as Penicillium rubrum (van Reenen-Hoekstra et al. 1990), T. rugulosus, T. verruculosus (Pitt 1979) and T. ramulosus (Visagie et al. 2009).

PCR reactions yielded amplicons of ± 600 and ± 500 bp in length for ITS and β-tubulin, respectively. The aligned ITS dataset (Fig. 1) was 611 base pairs long. The aligned β-tubulin datasets was 382 and 374 bp long, for the T. solicola clade (Fig. 1, Clade 1) and T. chloroloma / T. ptychoconidium clade (Fig. 1, Clade 2), respectively. Morphological identifications were confirmed by neighbour-joining analysis that resolved the fynbos strains in the larger clade containing Talaromyces spp. (Fig. 1). Sequenced strains from the three, presumed to be new species, clustered in three separate clades according to their respective morphological characters. The three groups could also be separate from previously described species, based on morphological differences, supporting their novelty.

Fig. 1

Neighbour-joining trees based on the ITS1–5.8S–ITS2 rDNA and β-tubulin (in blocks) gene regions, showing relationships within Talaromyces spp. and strains isolated from fynbos soil. Numbers at branching nodes represent bootstrap values (1 000 replicates), with bold branches indicating bootstrap values higher than 80 %. Talaromyces thermophilus was selected as outgroup for the ITS phylogeny. Penicillium lignorum and T. phialosporus was chosen as outgroup for the T. solicola clade (top block = Clade 1) phylogeny, with T. calidicanius, T. duclauxii, T. palmae and T. panamensis the outgroup for the T. chloroloma / T. ptychoconidium clade (bottom block = Clade 2) phylogeny.

One of the fynbos strains resembled P. minioluteum. Pitt’s 1979 description of this species was based on isolate FRR1714 (= IMI 89377, CBS 642.68) as ex-neotype. This isolate, however, more closely resembles P. rubrum sensu Raper & Thom and was renamed accordingly (van Reenen-Hoekstra et al. 1990). The fynbos isolates displayed characters typical of this species, growing faster than P. minioluteum, as well as being strongly funiculose and producing the greenish to red colony reverse. The phylogenetic placement of P. rubrum and P. minioluteum makes sense considering their morphological characters (Fig. 1). Penicillium minioluteum is resolved in a clade containing slow growing species on CYA, whereas P. rubrum is closely related to P. funiculosum, P. pinophilum and P. verruculosum which all grow more rapidly on CYA. Samson et al. (2011) did not transfer P. rubrum to Talaromyces since its taxonomy remains unresolved because no type material was located. A number of fynbos strains, however, did match sequences of strain FRR 1714, as well as the description provided by van Reenen-Hoekstra et al. (1990). These were thus identified as P. rubrum.

Fynbos strains identified as T. verruculosus, produced bright yellow mycelia on CYA and MEA, as well as spheroid, verrucose conidia, which are definitive characters for the species (Pitt 1979). Talaromyces rugulosus strains grew slowly on both CYA and MEA, and produce conidiophores that often are multi-ramulate, ending in acerose to almost ampulliform-like phialides. The recently described T. ramulosus (Visagie et al. 2009), characterised by strongly funiculose colonies, producing clear glutinous exudates and short synnema after prolonged incubation, was also isolated during the survey. Three of the fynbos taxa exhibited unique morphological characters that did not conform to descriptions of any known species. Based on these analyses, these are presumed to be new to science and are described below.

TAXONOMY

Visagie & K. Jacobs, sp. nov. — MycoBank MB564326; Fig. 2, 3

Fig. 2

Most important taxonomic characters of T. chloroloma, holotype (PREM 60033), distinguishing it from closely related species. a. Colonies of T. chloroloma incubated on CYA, MEA and YES from left to right (top row = obverse, bottom row = reverse); b, c. synnemata produced on CYA after prolonged incubation; d–g. conidiophores produced in culture; h. ellipsoidal, smooth-walled conidia. — Scale bars: b, c = 50 μm; in g = 10 μm, applies to d–h.

Fig. 3

Talaromyces chloroloma line drawings from holotype (PREM 60033) material. — Scale bar: 10 μm for a–c; 50 μm for d, e.

Etymology. Latin, chloroloma: chloros- = green + -loma = fringe. Indicating the green conidia en masse near fringes.

Colony morphology, CYA, 25 °C, 7 d: Colonies 34–37 mm diam, plane, moderately dense; texture floccose with funicles present, determinate synnemata produced in incidental sunlight after prolonged incubation; margins subsurface, irregular, mycelia white, 4–5 mm wide, characteristic spiral growth at edge of colonies; conidiogenesis medium to heavy, olive-brown (4e5–4e7) at centre, pastel green (27a4) at edge; exudate and soluble pigment absent, reverse greyish ruby (12c3–12c4) at centre, greenish white (26a2) elsewhere. At 5 °C, 7 d: Germination; 37 °C, 7 d: Colonies 9–12 mm diam, plane; moderately dense; texture floccose to loosely funiculose; white mycelia; conidiogenesis sparse to medium, dark green (25f7); exudate and soluble pigment absent, reverse olive (1e6). MEA, 25 °C, 7 d: Colonies 43–48 mm diam, plane, moderately dense; texture strongly funiculose to floccose; margins subsurface, 4–5 mm, irregular, mycelia white; conidiogenesis medium to dense, olive-brown (27a4) at centre, greyish green (25c6–25d6) elsewhere; exudate and soluble pigment absent, reverse greyish green (29d5). YES, 25 °C, 7 d: Colonies 39–42 mm diam, plane, moderately dense; texture strongly funiculose; margins wide, 3–5 mm, regular, mycelia white; conidiogenesis medium to heavy, dull green to greyish green (26E4–26E6); exudate and soluble pigment absent, reverse greyish yellow (2A3–2A4). G25N, 25 °C, 7 d: Micro-colonies.

Conidiophores borne from well-defined funicles, having an olive-like pigmentation, stipes smooth-walled, 12–45 × 3–4 μm when borne on funicles, and 700–1200 μm when borne in synnema, bearing terminal biverticillate penicilli, with terverticillate penicilli also present; branches, when present in appressed to almost parallel whorls of 2–4, but mostly 3 branches, 11–12(−15) × 3–3.5 μm; metulae cylindrical in whorls of 3–5, appressed, 7.5–9(−10.5) × 2–3 μm; phialides 4–5 per metula, closely appressed, acerose, 7–8(−8.5) × 1.5–2.5 μm, tapering to fine apical pore, 0.5–1 μm; conidia ellipsoidal, 2.5–3.5 × 1.5–2 μm, smooth-walled, borne in disordered chains, sparse larger conidia present 4.5–5(−6) × 2–2.5 μm; synnemata on CYA produced after 14–21 d of growth in incidental sunlight determinate, unbranched white stalk 700–1200 × 80–150 μm, 400–580 μm across apex, conidiophores bearing a powdery, dark green conidial mass at the apex.

Specimens examined. South Africa, Western Cape Province, Malmesbury, Riverlands S 33,49066°; E 18,58388°, isolated from soil, 21 Feb. 2007, collected by C.M. Visagie, holotype PREM 60033, culture ex-type CV2802 (KAS 4250, DAOM 241016); Western Cape Province, Malmesbury, Riverlands S 33,49066°; E 18,58388°, isolated from soil, 21 Feb. 2007, collected by C.M. Visagie, paratype PREM 60034, culture ex-type CV 2803 (KAS 4251).

Visagie & K. Jacobs, sp. nov. — MycoBank MB564327; Fig. 4, 5

Fig. 4

The most important taxonomic characters distinguishing T. ptychoconidium, holotype (PREM 60041), from closely related species. a. Colonies of T. ptychoconidium grown on CYA, MEA and YES from left to right (top row = obverse, bottom row = reverse); b. typical loosely funiculose texture seen on MEA, together with abundant exudate and yellow mycelia; c–g. conidiophores produced in culture; h. ellipsoidal, fusiform, spirally roughened conidia. — Scale bar: in g = 10 μm, applies to c–g.

Fig. 5

Talaromyces ptychoconidium line drawings from holotype (PREM 60041) material. — Scale bar: 10 μm for a–c; 50 μm for d.

Etymology. Latin, ptychoconidium: ptycho = ridge. Refer to the ridges on the conidia that are distinctive in this species.

Colony morphology, CYA, 25 °C, 7 d: Colonies 8–12 mm diam, plane, loose to moderately dense; texture floccose; margins subsurface, 3–4 mm wide, regular, mycelia white; conidiogenesis sometimes absent, mostly sparse, conidia en masse greyish green (1d4) when present; clear sticky exudate produced, soluble pigment absent, reverse pale to greyish yellow (1d4). At 5 °C, 7 d: No germination; 37 °C, 7 d: Colonies 8–9 mm diam, plane; texture velutinous; white mycelia; conidiogenesis sparse, brownish orange (6c3–6c5); exudate and soluble pigment absent, reverse pale (6b2). MEA, 25 °C, 7 d: Colonies 15–21 mm diam, plane, loose, sometimes having a somewhat pinkish colour; texture loosely funiculose; margins subsurface, narrow, irregular, mycelia yellow; conidiogenesis sparse to moderate, dark green (28f4); clear to pale slimy exudate produced, soluble pigment absent, reverse raw umber (5f8). YES, 25 °C, 7 d: Colonies 20–25 mm diam, moderately sulcate to umbonate; margins low, mycelia white, margins wide; conidiogenesis sparse to moderate, conidia en masse spinach green to dull green (29e3–29e6); yellow soluble pigment yellow (2a5–2a6), abundant yellow to golden exudate, reverse persian red (8e8) at centre, becoming light brown (6d7–6d8) nearer to edge. G25N, 25 °C, 7 d: Colonies 4–5 mm diam, plane; margins low, white mycelia; conidiogenesis absent; clear exudate produced, soluble pigment absent, reverse white.

Conidiophores borne from aerial hyphae and loose funicles, having a greenish pigment, stipes smooth-walled, (38−)60–93 × 2.5–3.5 μm when borne on aerial hyphae and funicles, bearing strictly terminal biverticillate penicilli; metulae cylindrical in whorls of (3−)5–7, appressed, 10–11.5(−12.5) × 2–3 μm; phialides 3–4 per metula, closely appressed, acerose, 10–12 × 2–2.5 μm, tapering to fine apical pore, 0.5–1 μm; conidia ellipsoidal, apiculate, 3–4.5(−5) × 2–3 μm, spirally rough-walled, borne in disordered chains.

Specimens examined. South Africa, Western Cape Province, Malmesbury, Riverlands S 33,49066°; E 18,58388°, isolated from soil, 21 Feb. 2007, collected by C.M. Visagie, holotype PREM 60041, culture ex-type CV 2808 (KAS 4245, DAOM 241017); Western Cape Province, Malmesbury, Riverlands S 33,49066°; E 18,58388°, isolated from soil, 21 Feb. 2007, collected by C.M. Visagie, paratype PREM 60043, culture ex-type CV 2806 (KAS 4246), paratype PREM 60042, culture ex-type CV 2807 (KAS 4247).

Visagie & K. Jacobs, sp. nov. — MycoBank MB564328; Fig. 6, 7

Fig. 6

Talaromyces solicola line drawings from holotype (PREM 60037) material. — Scale bar: 10 μm for a–c; 50 μm for d.

Fig. 7

The most important taxonomic characters distinguishing T. solicola, holotype (PREM 60037), from closely related species. a. Colonies of T. solicola grown on CYA, MEA and YES from left to right (top row = obverse, bottom row = reverse); b. floccose to velutinous colony texture on MEA; c–g. conidiophores produced in culture; h. subspheroidal, rough-walled verrucose conidia. — Scale bar: in g = 10 μm, applies to c–g.

Etymology. Latin, solicola: solum = soil; -cola = an inhabitant, an inhabitant of soil.

Colony morphology, CYA, 25 °C, 7 d: Colonies 3 mm diam, sometimes only germination, low, plane, loose; texture floccose; margins low, irregular, mycelia white; conidiogenesis absent to sparse, conidia en masse white when present; clear to orange exudate produced, soluble pigment absent, reverse pale white. At 5 °C, 7 d: No germination. 37 °C, 7 d: No germination. MEA, 25 °C, 7 d: Colonies 20–21 mm diam, sometimes up to 25 mm, low, sulcate and sometimes slightly sunken at centre, loose, sometimes having a brownish orange colour; texture floccose to velutinous; margins low, 3–4 mm wide, regular, mycelia white; conidiogenesis medium, dark green (28f4); clear exudate produced, soluble pigment absent, reverse colouration brownish grey (6d2) at centre and greyish green (1c3) elsewhere. YES, 25 °C, 7 d: Colonies 8–10 mm diam, dense; texture floccose; margins low, irregular, mycelia white; conidiogenesis sparse to moderate, conidia en masse greyish green, but colour rather distorted because of reddish pink exudates produced; soluble pigment absent, reverse dark brown (8F6–8F8). G25N, 25 °C, 7 d: No germination or growth.

Conidiophores borne from aerial hyphae, stipes smooth-walled, (90−)160–230 × 2.5–3.5 μm, bearing strictly terminal biverticillate penicilli; metulae cylindrical in whorls of 5–8 sometimes up to 10, appressed, 8.5–10(−11) × 2.5–3.5 μm; phialides 3–4 per metula, closely appressed, acerose 9–11 × 2–2.5 μm, tapering to fine apical pore, 0.5–1 μm; conidia subspheroidal, verrucose, (2−)2.5–3.5 × 2–2.5 μm, rough-walled, borne in disordered chains.

Specimens examined. South Africa, Western Cape Province, Malmesbury, Riverlands S 33,49066°; E 18,58388°, isolated from soil, 21 Feb. 2007, collected by C.M. Visagie, holotype PREM 60037, culture ex-type CV 2800 (KAS 4235, DAOM 241015); Western Cape Province, Malmesbury, Riverlands S 33,49066°; E 18,58388°, isolated from soil, 21 Feb. 2007, collected by C.M. Visagie, paratype PREM 60038, culture ex-type CV 2801 (KAS 4236).

1. Colonies on CYA < 5 mm ........................ 2

1. Colonies on CYA > 5 mm ........................ 4

2. Conidia distinctly rough-walled ............ T. solicola

2. Conidia smooth-walled .......................... 3

3. Growth only on acidified (pH 5/less) CYA; stipes < 100 μm ..................................... P. lignorum

3. Colonies on CYA 2–4 mm, sometimes only microcolony; stipes > 100 μm ......................... T. diversus

4. Conidia ornamented with transverse/spiral striations ... 5

4. Conidia without striations ......................... 6

5. Mycelia red on CYA and MEA; stipes vesiculate ........................................... T. purpureus

5. Mycelia white on CYA and MEA; stipes non-vesiculate .................................. T. ptychoconidium

6. Colonies on CYA < 12 mm and MEA < 20 mm ........ 7

6. Colonies on CYA > 12 mm or MEA > 20 mm ......... 8

7. Red colony pigments commonly produced on CYA and MEA; Metulae borne on vesiculated stipes ..... T. erythromellis

7. No red colony pigmentation; stipes non-vesiculated .......................................... T. rugulosus

8. Synnemata never produced ...................... 9

8. Synnemata produce, mostly after prolonged incubation 10

9. Colonies on CYA > 20 mm, conidia rough-walled ........................................ T. purpurogenus

9. Colonies on CYA < 20 mm, conidia smooth-walled ................................... T. minioluteus

10. Synnemata stalks and mycelia on CYA and MEA pigmented ........................................ 11

10. Synnemata stalks and mycelia white ............. 12

11. Determinate synnemata > 2000 μm ..... T. dendriticus

11. Indeterminate synnemata < 2000 μm ........................................... T. pseudostromaticus

12. Conidia en masse on CYA pink to greyish green; synnemata 100–250 μm tall; abundant sticky exudates produced ................................... T. ramulosus

12. Synnemata > 250 μm ......................... 13

13. Colonies on CYA 34–37 mm and MEA 43–48 mm; conidia en masse olive brown, with light green edge ......................... T. chloroloma

13. Colonies on CYA 19–31 mm and MEA 32–40 mm; conidia en masse greyish green to greyish turquoise ............................................ T. cecidicola

KEY TO THE NEWLY DESCRIBED TALAROMYCES SPECIES AND THE CLOSELY RELATED SISTER TAXA

* Species described in this paper

DISCUSSION

Talaromyces is distinguished from closely related genera by its cleistothecial ascomata that have a soft hyphal exterior giving it a yellow, cream, pink or reddish colour (Samson et al. 2011). In its anamorph state, typical symmetric biverticillate, sometimes terverticillate, conidiophores are produced that bear thin acerose phialides. A few species do produce an ampulliform-like phialide with a thin, long neck (Pitt 1979, Samson et al. 2011). Species typically have a metula to phialide length ratio of ± 1–1.2, as well as poor growth (Pitt 1979). Seven taxa isolated from Atlantis sand fynbos soil conform to these characters, although no ascomata was observed, and was subsequently placed in Talaromyces. Species isolated included Penicillium rubrum, T. ramulosus T. rugulosus-like strains and T. verruculosus, together with the three species described here (Fig. 1).

In a similar study, Allsopp et al. (1987) recorded P. verruculosum (= T. verruculosus), P. purpurogenum (= T. purpurogenus) and P. funiculosum (= T. funiculosus) from soil collected at Riverlands Nature Reserve. Thus, they only isolated three species from the Talaromyces clade, compared to the eight isolated from this study. This may, however, be due to the fact that we mostly found this group to occur in low numbers and often in only one soil sample. In their study, Allsopp et al. (1987) included only species that occurred in 10 or more samples, which may explain the low species diversity reported for this habitat. This seems to hold true for new isolations made from additional fynbos biotypes. In a current study, a large number of strains representing Talaromyces were isolated. Once again these also seem to be habitat specific and occur in low numbers. Reasons for this species distribution remain unclear, although the heterogeneous nature of the fynbos, with its high beta-diversity (Goldblatt & Manning 2002), may play a role. In addition, we believe that some of these species might have alternative dispersal strategies, opposed to air dispersal, and have associations with specific plant species occurring in the fynbos (Visagie et al. 2009).

This idea was also voiced by Pitt (1979), who mentioned species with possible narrow spectrum habitats and alternative dispersal strategies. Also, new species from this group are often isolated from very specific habitats. For example, Talaromyces cecidicola, was isolated and described from wasp galls on Quercus pacifica trees (Seifert et al. 2004), T. dendriticus always seems to be associated with Eucalyptus (Pitt 1979, Seifert et al. 2004), T. pseudostromaticus was isolated from various bird species in Minnesota (Hodges et al. 1970), and P. aureocephalum is currently only known to occur on dried-out leaf litter from Barcelona (Muntañola-Cvetković et al. 2001, Llimona et al. 2006). Distribution patterns and dispersal methods in Penicillium and Talaromyces will be investigated in future studies.

One of the species isolated from fynbos soils, conformed to Pitt’s (1979) description for T. rugulosus. The description of this slow-growing species is, however, considered too broad. For instance, conidia were described as smooth to verrucose. This allows for a broad spectrum of morphological variation within the taxon. Comparisons of the ITS gene region showed that the fynbos strains form a distinct clade next to previously sequenced P. rugulosum strains. In addition to this, a morphologically distinct species, also isolated during this study, forms a clade within the larger P. rugulosum group. β-tubulin sequences did, however, not resolve these into well-defined clades. Without a more detailed sequence dataset of T. rugulosus, its previously designated synonyms and close relatives, this issue cannot be resolved. We, therefore, consider T. rugulosus to possibly represent a species complex and this needs to be addressed in future studies.

Talaromyces chloroloma is characterised by strongly funiculose colonies producing olive coloured conidia en masse. Determinate synnemata are produced on CYA after 2 wk of incubation. The species is further characterised by appressed olive-coloured conidiophores, borne on short stipes. Talaromyces chloroloma is morphologically similar to other synnemata producers, T. cecidicola, T. dendriticus and T. ramulosus. The new species is easily distinguished from T. cecidicola by faster growth on CYA (34–37 vs 19–31 mm) and MEA (43–48 vs 32–40 mm) at 25 °C and its ability to grow at 37 °C on CYA. In addition, T. chlo- roloma conidiophores have shorter stipes (12–45 vs 20–80 μm), but generally, longer synnemata (700–1200 vs 250–1250 μm) compared to T. cecidicola. The longer synnemata produced by T. chloroloma also distinguishes it from T. ramulosus which has very short synnemata (110–150 μm) produced on MEA. Conidial structures in T. chloroloma are also much denser and conidia have an olive-brown colour compared to the pink to greyish green conidia of T. ramulosus. Talaromyces dendriticus is easily distinguished by its production of long (2–4 mm) synnemata with yellow stalks, compared to T. chloroloma that has shorter synnemata and white stalks. The morphological relationship of these species are reflected in their phylogeny and are sister taxa with well-supported branches in a neighbour-joining tree (Fig. 1).

Talaromyces ptychoconidium characteristically displays restricted growth on CYA and MEA. Its most striking feature is the closely appressed conidiophores producing spirally rough-walled ellipsoid conidia (Fig. 4, 5). The phylogenetic analysis suggests an affinity to T. purpureus as its sister taxon. Interestingly, both of these species produce spirally ornamented conidia. These two species are readily distinguished from each other, based on the red mycelia produced by T. purpureus compared to the white mycelia of T. ptychoconidium. Microscopically, T. purpureus has vesiculate stipes with both metulae (7–10 μm) and phialides (6–10 μm) shorter than that of T. ptychoconidium.

The third species, T. solicola, is distinguished by its characteristic poor growth on CYA. Its phylogenetic placement resolves T. solicola in a clade where species all display similar restricted growth. Talaromyces erythromellis, T. diversus, T. purpurogenus and P. lignorum all display similarly poor growth on CYA and MEA, the latter species only growing on acidified CYA (Pitt 1979). Talaromyces solicola is easily distinguished from all close relatives based on its heavy rough-walled, spheroidal to subspheroidal conidia, compared to the smooth-walled conidia of its sister taxa. In addition to the texture of its conidial walls, P. lignorum produces shorter stipes (15–50(−100) μm). Talaromyces diversus often produces yellow coloured mycelia at colony peripheral zones, which are absent in T. solicola. On MEA, T. erythromellis grows much faster than the new species. Phylogenetic analysis of both the ITS and β-tubulin genes confirmed the novelty of T. solicola, forming well-defined clades separate from any close relatives.

Penicillium and Talaromyces in general are regarded to have soil as its principle habitat, with a large proportion of species only known from soil (Pitt 1979). Fynbos soil is considered an especially harsh environment because of its acidity and poor nutrient levels (Kruger et al. 1983), which is worsened by the low rainfall and high temperatures during summer and high rainfall and low temperatures during winter (Richards et al. 1997). Other external factors influencing organisms that live in the fynbos is the heterogeneity of the soil and plants, as well as the constant fires associated with the habitat (Goldblatt & Manning 2002). These are just some factors that affect and place constant evolutionary pressure on organisms that actively live in this habitat. It is, however, of interest to note that the species isolated in this study does not seem to be confined to the soil environment. Talaromyces ramulosus has been isolated from Protea infructescences (Visagie et al. 2008) and together with a number of other Penicillium and Talaromyces spp. were also isolated from mites in these infructescences. Talaromyces ramulosum and T. chloroloma were also isolated from apple orchards in the Western Cape (van der Walt et al. 2010). The occurrence and distribution pattern of species occurring in fynbos from the Western Cape are still unknown. With more studies like these, uncovering and describing species from this unique area, which has diverse fungal communities, might lead to surprising discoveries related to their distribution and ecology.