377

Alberto M. STCHIGEL1, Josep CANO1, Walter MAC CORMACK2 and Josep GUARRO1

"Unitat de Microbiologia, Facultat de Medicina i Cie[ ncies de la Salut & Institut d’Estudis Avançats, Universitat Rovira i Virgili, C}Sant Llorenç.

21, 43201 Reus, Spain

# Instituto AntaU rtico Argentino, Departamento de BiologıUa, C}Cerrito 1248 (1010), Buenos Aires, Argentina.

E-mail : umb!fmcs.urv.es

Received 12 November 1999 ; accepted 4 July 2000.

A new ascomycete, Antarctomyces psychrotrophicus gen. et sp. nov., characterised by naked asci, hyaline, thick-walled, ellipsoidal to

fusiform, echinulate ascospores, and blastoconidia, isolated from Antarctican soil samples, is described and illustrated. Analysis of the

nuclear rDNA ITS region sequences showed that this taxon is related to Thelebolaceae.

INTRODUCTION

During the summer expedition of the ‘ Instituto Anta! rticoArgentino ’ to the Antarctica, W.M.C. collected soil samples

near the ‘ Jubany ’ Argentinian base (King George Island,

South Shetland Islands). Two strains of an undescribed

ascomycete were isolated in axenic culture. These were

characterised by rudimentary ascomata, composed of a cluster

of a small number of asci without a peridium, and hyaline,

ellipsoidal to fusiform, spinulose ascospores. It was difficult to

determine its taxonomic placement from morphological

characteristics alone, so we compared the sequence of its ITS

region with those of other morphologically similar fungi of

uncertain taxonomic position, and also representatives of

Eurotiales, Onygenales, Pezizales and Sordariales.

MATERIALS AND METHODS

Fungal isolation

Soil samples were collected near the ‘ Jubany ’ Argentinian

base (62° 14« S, 58° 40« W) on King George Island, South

Shetland Islands, Antarctica. The terrain is basically basaltic

and metamorphosed rocks, and penguin dung is very common.

The vegetation is mainly algae as Prasiola crispa, lichens

(including Acarospora molybdina, Lecidea auriculata, Caloplaca

spp.), mosses (Andreaea depressinervis, A. regularis, Brachytecium

antarcticum, Bryum dichotomum, Grimnia antartici, Hypnum

sarmentosum, Pogonatum alpinum, Tortula excelsa, etc.), and the

plants Colobanthus quitensis, Deschampsia antarctica and Poa

pratensis (Cabrera 1994, Lindsay 1971, Mo$ ller & Dreyfuss

1996). The following climatic data were reported for 1995 :

average temperature ®1±5 °C, minimum of ®19±9 ° and

maximum of 10±4 ° ; total annual precipitation 273 mm, and

total annual snowfall 1257 cm; and the average humidity was

88%. Material was collected mainly from the A horizon,

placed into sterilised polyethylene bags closed by rubber

band, and stored in a refrigerator at ®20 °.Fungal isolation was by the soil plate method (Warcup

1950) in which suspensions were cultured on potato carrot

agar with 30 mg l−" chloramphenicol (PCA; potatoes, 20 g ;

carrot, 20 g ; agar, 20 g ; tap water, 1 l). We also used a

modification of Furuya and Naito’s method (1979). About

1 g of soil was suspended in 5 ml of 5% v}v acetic acid, shaken

vigorously for 5 min, and left for a further 5 min. The layer

of acetic acid was decanted, the residual soil resuspended with

9 ml of sterilised water, and the suspensions plated in a Petri

dish. PCA with chloramphenicol was placed on top of the soil

suspension and mixed. All cultures were incubated at 11–12°under 12 h of darkness, alternating with 12 h of cool white

fluorescent light.

The strains were grown on oatmeal agar (OA; Difco), PCA,

potato dextrose agar (PDA; Difco) and malt extract agar

(MEA; Difco) at room temperature (22–25 °), 11–12 °, and

4–6 ° under 12 h of darkness, alternating with 12 h of cool

white fluorescent light. Colour notations in parentheses are

from Kornerup & Wanscher (1984). Structures were measured

in lactophenol.

Molecular study

Table 1 lists the strains used in the study. The sequences

obtained from Momol & Kimbrough (1994) are not available

in any DNA sequences database checked. Monascus purpureus,

Neurospora crassa and Talaromyces flavus var. macrosporus were

obtained from EMBL. New sequences were obtained for

Amauroascus niger, Amauroascus volatilis-patellus, Antarctomyces

psychrotrophicus, Aphanoascus keratinophylus, Calyptrozyma

Mycol. Res. 105 (3) : 377–382 (March 2001). Printed in the United Kingdom.

Antarctomyces psychrotrophicus gen. et sp. nov., a newascomycete from Antarctica

Antarctomyces psychrotrophicus gen. et sp. nov. 378

Table 1. List of strains, sources and sequences used in the analysis.

Species Strain Origin

EMBL accession

numbers

Amauroascus niger IFO 32599 Soil AJ 133434

Amauroascus volatilis-patellus UAMH 3406 Soil AJ 133435

Antarctomyces psychrotrophicus FMR 6368 Soil AJ 133431

Aphanoascus keratinophylus IMI 319010 Soil AJ 133436

Ascodesmis nigricans* FLAS 122 Soil —

Ascodesmis sphaerospora* FLAS 260 Rat dung —

Calyptrozyma arxii CBS 354.92 Human oesophagus AJ 133432

Eleutherascus lectardii* FLAS 300 Salty soil —

Lamprospora sp.* FLAS 346 Soil —

Monascella botryosa CBS 233.85 Soil AJ 133433

Monascus purpureus ATCC 16365 — U18356

Neurospora crassa — — M13906

Pyronema domesticum* ATCC 14881 Steamed soil —

Saccobolus depauperatus* FLAS 106 Cow dung —

Talaromyces flavus var. macrosporus FRR 2386 — U18354

Thelebolus sp.* IMI 67944 Dung —

* Sequences from Momol & Kimbrough (1994). ATCC, American Type Culture Collection ; CBS, Centralbureau voor Schimmelcultures; FLAS, Florida

Agricultural Experiment Station culture collection ; FMR, Facultat de Medicina de Reus culture collection ; FRR, CSIRO Food Research Laboratory ; IFO, Institute

of Fermentation of Osaka ; IMI, CABI Bioscience UK Centre ; UAMH, University of Alberta Microfungus Collection and Herbarium.

arxii, and Monascella botryosa. The DNA was isolated as

described by Estruch et al. (1989) with some modifications

(Guillamo! n et al. 1996). The strains were grown at 20 °C in

Sabouraud broth in Ehrlenmeyer flasks and shaken at 200 rpm.

The mycelium was collected by filtration through nytal mesh

(42 µm pore size), washed with distilled water, blotted with

paper towels, frozen with liquid nitrogen and ground to a fine

powder with a mortar and pestle. The powder was incubated

for 1 h at 65° in 2 ml of extraction buffer 80±2 (Tris–HCl

pH 8±0, 0±25 NaCl, 25 m EDTA, 0±5% SDS). The lysate

was extracted with phenol-chloroform-isoamyl alcohol

solution (25 :24 :1) and DNA was recovered by isopropanol

precipitation. The pellet was washed with 70% v}v ethanol,

dried under vacuum and resuspended in TE buffer (10 m

Tris–HCl pH 8±0, EDTA 1 m).

The rDNA ITS region containing ITS1 and ITS2 and the

intervening 5±8 S rRNA gene were amplified as described by

Gene! et al. (1996), using a Perkin–Elmer 2400 thermal cycler

(Perkin–Elmer Cetus corporation, Emeryville, CA). Primers

ITS5 (5«-GGAAGTAAAAGTCGTAACAAGG-3«) and ITS4

(5«-TCCTCCGCTTATTGATATGC-3«) (White et al. 1990)

were used. The amplification program consisted of pre-

denaturalisation at 94–6 ° for 5 min, 30 cycles at 95 ° for 30 s,

50 ° for 1 min and 72 ° for 1 min, and final incubation at

72 ° for 7 min to complete the final extension. The final

products were resolved by electrophoresis in a 2% agarose

MP gel (Boehringer–Mannheim), and cleaned following the

GENECLEAN II protocol (BIO 101). The molecular weights

of amplified DNA were estimated by comparing them with

100 bp DNA leader (Gibco–BRL) standard lane.

The protocol ‘Taq DyeDeoxy Terminator Cycle

Sequencing Kit ’ (Applied Biosystems, Gouda) was used for

sequencing. Reactions were performed using the primers ITS5

and ITS4 (White et al. 1990) and run on a 310 DNA sequencer

(Applied Biosystems). The new sequences were aligned using

the Clustal W, version 1±5, computer program for multiple

sequence alignment (Thompson et al. 1994). Cladistic analyses

using the neighbour-joining method (Saitou & Nei 1987) and

parsimony were performed with the MEGA 1.0 computer

program (Kumar et al. 1993). Confidence values for individual

branches were determined by bootstrap analyses (1000

pseudoreplicates). Nucleotide composition, frequencies from

pairwise comparisons and alignment gap sequences were

performed with the MEGA 1.0 computer program.

RESULTS AND DISCUSSION

Taxonomy

Antarctomyces Stchigel & Guarro, gen. nov

Mycelium ex hyphis septatis, ramosis vel simplicibus, anastomo-

santibus, hyalinis compositum. Ascomata ex ascis nudis composita,

sine excipulo. Asci ellipsoidei vel subglobosi, unitunicati, non-

catenati, octospori. Paraphyses nullae. Ascosporae ellipsoideae vel

fusiformes, hyalinae, spinulosae, sine poro germinali, unicellulares.

Typus : Antarctomyces psychrotrophicus Stchigel & Guarro.

Mycelium mainly submerged, composed of septate, branched

and unbranched, anastomosing, hyaline hyphae. Ascomata

composed of naked asci, without excipulum. Asci ellipsoidal to

subglobose, unitunicate, non-catenate, 8-spored. Paraphyses

absent. Ascospores ellipsoidal to fusiform, hyaline, spinulose,

without germ pores, 1-celled.

Antarctomyces psychrotrophicus Stchigel & Guarro, sp.nov. (Figs 1–15)

Mycelium ex hyphis hyalinis, ramosis vel simplicibus anasto-

mosantibus, septatis, (1–)4–7 µm diam compositum; hyphae

tenuitunicatae vel crassitunicatae. Coloniae in agaro cum decocto

tuberorum et carotarum (PCA) planae, tenues, hyalinae. Ascomata

e hyphis initialibus duabus involutis formantia. Ascomata ex

ascis nudis composita, 2–7 in numero, sine excipulo. Asci

15–19¬12–13 µm, ellipsoidei vel subglobosi, non-estipitati, uni-

tunicati, crassitunicati, non-catenati, octospori. Paraphyses nullae.

A. M. Stchigel and others 379

1

2

5

6

7

8 10

9

3 4

Fig. 1–10. Antarctomyces psychrotrophicus. Fig. 1. Ascomata initials. Fig. 2. Ascomata initials forming a crozier. Fig. 3. Cluster of asci

containing the ascospores. Fig. 4. Free ascospores. Note the spinulose surface and the thick ascospore wall. Figs 5–7. Sporothrix-like

anamorph. Fig. 8. View of a funicle formed at 12 °C on PDA. Fig. 9. Ascus and ascospores. Fig. 10. Ascospore showing the spinose

outer wall. Bar Figs 1–2, 5–8¯ 20 µm; Figs 3–4¯ 12±5 µm; Fig. 9¯ 10 µm; Fig. 10¯ 1 µm.

Antarctomyces psychrotrophicus gen. et sp. nov. 380

11

13

12

14

15

Figs 11–15. Antarctomyces psychrotrophicus. Figs 11, 12. Ascomatal initials forms. Fig. 13. Cluster of thick-walled asci, showing young

and mature ascospores inside. Fig. 14. Sporothrix-like anamorph. Fig. 15. Conidia of different size. Bar¯ 15 µm.

Ascosporae 7–10¬4–5±5 µm, ellipsoideae vel fusiformes, hyalinae,

spinulosae, sine poro germinali, unicellulares. Anamorphosis

blastoconidiis.

Typus : Antarctica : South Shetland Islands, King George Island,

ex solo, 10 Nov 1996, W. Mac Cormack [isol. A. M. Stchigel] (IMI

378528 – holotypus, FMR 6368 – isotypus).

A. M. Stchigel and others 381

Amauroascus niger

Amauroascus volatilis-patellus

Aphanoascus keratinophylus

Monascus purpureus

Talaromyces flavus var. macrosporus

Calyptrozyma arxii

Thelebolus sp.

Antarctomyces psychrotrophicus

Monascella botryosa

Lamprospora sp.

Pyronema domesticum

Eleutherascus lectardii

Saccobolus depauperatus

Ascodesmis nigricans

Ascodesmis sphaerospora

Neurospora crassa

99

100

100

100

100

100

97

56

100

100

69

Fig. 16. Neighbour-joining phylogenetic tree of the aligned sequences of the studied strains. Confidence limits of branches (indicated in

% along the branches) were created in a bootstrap analysis using 500 trials. Bar¯ 0±68% sequence divergence.

Mycelium mainly submerged, composed of hyaline, branched

and unbranched, anastomosing, septate hyphae ; hyphae

(1–)4–7 µm broad, thin to thick-walled. Colonies on PCA

33–47 mm diam in 14 d at 22–25 °C, plane, thin, white, with

irregular margins ; reverse uncoloured. Ascomatal initials begin

to develop from the coiling of two side branches, occasionally

disposed in tandem. Ascomata composed of naked asci, single

or in groups of 2–7, arising directly from the fertile hyphae,

without an exciple. Paraphyses absent. Asci 15–19¬12–13 µm,

subglobose to ellipsoidal, non-stipitate, unitunicate, thick-

walled, non-catenate, 8-spored, developed form croziers.

Ascospores 7–10¬4–5±5 µm, ellipsoidal to fusiform, hyaline,

spinulose, thick-walled, without germ pores, one-celled ; spines

c. 0±5 µm long.

Anamorph : Conidiophores 4–7 µm thick, hyaline, with lateral

cylindrical protuberances measuring 2–5¬1–2 µm. Conidio-

genous cells enteroblastic, integrated, intercalarly, determinate.

Conidia 3–20¬2–5 µm, subglobose to irregularly cylindrical,

hyaline, smooth, thick-walled, aggregated in slimy masses,

one-celled. Chlamydospores 10–15¬5–8 µm, irregular, single

or forming long chains, one or two-celled.

Colonies on PCA 32–43 mm diam after 14 d at 11–12 °C,

and 26–28 mm diam at 4–6 °, plane, thin, zonate, vegetative

mycelium mainly submerged, uncoloured ; reverse uncoloured.

Asci and chlamydospores abundant ; conidia absent.

Colonies on PDA 65–71 mm diam in 14 d at 22–25°, plane,thin, margins irregular, vegetative mycelium mainly sub-

merged, uncoloured ; reverse uncoloured. Chlamydospores

present, in long chains ; asci absent ; conidia present. At 11–12°the colonies attain 62–68 mm diam in 14 d, and 35–37 mm

diam at 4–6 °, plane, thin, margins irregular, vegetative

mycelium mainly submerged, dull blue (M 23D4), light blue

funicles in the central area, composed of sterile hyphae ;

reverse with the same colour. Asci abundant in the marginal

area ; chlamydospores in long chains ; conidia absent.

Colonies on OA 50–55 mm diam in 14 d at 22–25 °, plane,thin, margins fimbriate, vegetative mycelium mainly sub-

merged, uncoloured ; reverse uncoloured. Chlamydospores very

abundant, in long chains ; asci absent ; conidia present. At 12°the colonies 35–45 mm diam and at 4–6 ° 26–29 mm diam in

14 d, plane, thin, vegetative mycelium mainly submerged,

uncoloured ; reverse uncoloured. Asci and chlamydospores

abundant ; conidia absent.

Colonies on MEA 54–58 mm diam in 14 d at 22–25 °, with

the same cultural characteristics as in OA. Moniliform

mycelium present ; asci and chlamydospores absent ; conidia

absent. At 12 ° the colonies attaining a 45–49 mm diam in

14 d and 20–22 mm at 4–6 °, plane, thin, with the vegetative

mycelium mainly submerged, uncoloured ; reverse uncoloured.

Moniliform mycelium present ; asci and chlamydospores absent ;

conidia absent.

The main features of the A. psychrotrophicus ITS1–2 and 5±8S rDNA region sequence are : 547 bp ; 130 A; 131 C; 130 G

and 156 T. The location ITS1 from nucleotide 33 to 192,

the gene 5±8S rRNA from nucleotide 193 to 364 and the

location ITS2 from nucleotide 365 to 499.

Two other ascomycetes with simple sexual structures

consisting of clusters of a few asci with no peridium or exciple

and with more or less ellipsoidal and hyaline ascospores are

Calyptrozyma arxii Boekhout & Spaay 1995 and Monascella

Antarctomyces psychrotrophicus gen. et sp. nov. 382

botryosa Guarro & Arx 1986, each of which is monotypic. The

latter was isolated from Spanish soil (Guarro & Arx 1986) and

accommodated in the Onygenaceae (Hawksworth et al. 1995).

No anamorph has been observed in nature in this species.

Calyptrozyma was isolated from a human oesophagus in the

USA (Boekhout et al. 1995) and provisionally placed in

Eurotiales, though not in any family (Eriksson & Hawksworth

1996). The three taxa are distinguished mainly by their

anamorphs : blastoconidia in Antarctomyces : aleurio-, arthro-

and blastoconidia in Calyptrozyma ; and absent in Monascella.

The ascomatal initials are erect ascogonia surrounded by

coiled antheridia in M. botryosa, aggregations of generative

hyphae in C. arxii, and clustered antheridia and ascogonia in

A. psychrotrophicus ; the asci are clavate to obovate in M.

botryosa, cylindrical in C. arxii, and spherical to subspherical in

A. psychrotrophicus ; and the ornamentation of the ascospores

which are smooth-walled in C. arxii and in M. botryosa, and

spinulose in A. psychrotrophicus. Differences in the ITS-rRNA

gene sequences of these three species confirmed their

placement in different genera.

To infer the phylogenetic relationships of these taxa with

other morphologically similar ones and to establish a more

precise taxonomic position, we compared their ITS sequences

with those of 13 other species, some of which were obtained

from the EMBL. We chose representatives of Thelebolaceae

(Thelebolus sp.), Eurotiales (Monascus ruber and Talaromyces

flavus), Onygenales (Amauroascus niger, A. volatilis-patellis, and

Aphanoascus keratinophylus), Pezizales (Ascodesmis nigricans, A.

sphaerospora, Eleutherascus lectardii, Lamprospora sp., Pyronema

domesticum, and Saccobolus depauperatus), and Sordariales

(Neurospora crassa). The phylogenetic tree, based on analyses

of the ITS-5±8 rRNA gene sequences of all taxa studied with

the neighbour-joining method, demonstrated that Antarcto-

myces psychrotrophicus, Calyptrozyma arxii and Monascella

botryosa were not closely related phylogenetically. These

analyses showed the existence of two well-supported clades

(Fig. 16). The first clade, supported by a bootstrap value of

56% encompasses the Onygenales, Eurotiales, and a sister

subclade of Antarctomyces psychrotrophicus, Calyptrozyma arxii

and Thelebolus sp. (with a bootstrap value of 100%). In this

case C. arxii may be the ancestor of the other two taxa.

However, it is very difficult to establish any morphological

relationship among these tree taxa. Thelebolus and Antarcto-

myces share only thick-walled asci and ellipsoidal to fusiform,

thick-walled ascospores, which do not have germ pores. The

second clade, supported by a bootstrap value of 100%, is

formed by the pezizalean fungi ; Monascella botryosa was also

included in this group. Surprisingly, it did not cluster with the

species with simpler ascomatal structures, such as Ascodesmis

spp., Eleutherascus lectardii, and Saccobolus depauperatus, but

with Lamprospora sp. and Pyronema domesticum (Pyronemataceae)

whose ascomata are more developed. Kimbrough (1989)

pointed out a close relationship of somemembers of Onygenales

with simple structures and naked asci such as Amauroascus

with the Pezizales. He included this genus, together with

Ascodesmis and Eleutherascus, in the Ascodesmidaceae (Pezizales).

On the basis of the ascus structure, we disagreed that there was

such a relationship between Pezizales and Onygenales (Guarro

et al. 1992). In this study the two species of Amauroascus were

placed in a different clade from the Pezizales, which confirms

our previous opinion.

ACKNOWLEDGEMENTS

This work was supported by grant PM95-0160 from CICYT (Ministerio de

Educacio! n y Ciencia) and the Fundacio! Cie' ncia i Salut, Reus, Spain. The

authors are indebted to the Instituto Anta! rtico Argentino (IAA) for helping

to obtain samples. The senior author is grateful for the fellowship grant of the

Universitat Rovira i Virgili (U.R.V.), Catalonia, Spain.

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Corresponding Editor : R. S. Currah