975

Alberto M. STCHIGEL, Lidia FIGUERA, Jose! CANO and Josep GUARRO

Unitat de Microbiologia, Facultat de Medicina i Cie[ ncies de la Salut, Universitat Rovira i Virgili, C}Sant Llorencn 21,

43201 Reus, Spain.

E-mail : umb!astor.urv.es

Received 5 December 2001; accepted 18 June 2002.

Two new thermotolerant species of Thielavia were isolated from Easter Island and India, after treating soil samples

with 65% (v}v) ethyl alcohol : T. intermedia sp. nov. characterized by large, obovate, dark brown ascospores with a

terminal germ pore; and T. arxii sp. nov. distinguished by the large, ellipsoidal to fusiform, brown ascospores with a

prominent, lateral to medial germ pore. A phylogenetic study of the ITS-5.8S rDNA sequences of 18 species of

Thielavia and Melanocarpus thermophilus was performed. This analysis revealed that T. intermedia could be the

ancestor of the remaining species and that all the species tested were genetically closely related.

INTRODUCTION

Thielavia (Chaetomiaceae, Sordariales) is characterized

by spherical, non-ostiolate ascomata, with a thin

peridium, and one-celled, darkly pigmented ascospores.

Revisions of this genus have been proposed by Booth

(1961), Mouchacca (1973), Malloch & Cain (1973), von

Arx (1975), and von Arx, Figueras & Guarro (1988).

Von Arx (1973) excluded T. sepedonium and similar

species with ascospores with two germ pores and

Myceliophthora anamorphs, and erected the genus

Corynascus to accommodate these species. The last,

most comprehensive, revision by von Arx et al. (1988)

accepted 14 species. Subsequently, two more species

have been described (Chen & Chen 1996, Ito, Okane &

Nakagiri 1998). Here we describe two new species

recently isolated from Easter Island and Indian soils

respectively, and compare them molecularly with other

species of the genus.

MATERIALS AND METHODS

Collection and specimen isolation

Indian soil samples were collected close to Jaipur,

Rajasthan State, a tropical semiarid region dominated

by a hot climate ; the average temperature is 23 °C in

winter and 35 ° in summer, and the total annual

precipitation about 900 mm. Vegetation is mainly

grasses and shrubs. Easter Island is a triangular,

volcanic, semiarid island of 162 km# located in the

middle of the Pacific Ocean. Soil samples were collected

near Hanga-Roa city. The vegetation is mainly grasses,

with reduced forest masses composed of a few imported

trees, such as Eucalyptus spp. and Melia azederach. The

area is dominated by a subtropical maritime climate ;

the average temperature is 17±8 ° in winter and 23±7 ° in

summer. The total annual precipitation is about

1140 mm, with no dry season.

Collections were taken mainly from the superficial

layer of soil with sterilized polyethylene bags. These

were closed with rubber bands and labelled. On their

arrival at the laboratory the materials were stored at

4–7 ° until used. Approx. 1 g of soil was treated with

65% (v}v) ethyl alcohol according to Warcup & Baker

(1963). The suspensions were cultured on tap water

agar (TWA; agar, 20 g; tap water, 1000 ml) and

incubated at 42 ° under 12 h darkness alternating with

12 h cool white fluorescent light.

Cultural and morphological characterization

The morphological study was performed by growing

the isolates on oatmeal agar (OA; Difco), potato carrot

agar (PCA; potatoes, 20 g; carrot, 20 g; agar, 20 g; tap

water, 1000 ml), potato dextrose agar (PDA; Difco)

and malt extract agar (MEA; Difco) at 15 °, room

temperature (22–25 °), 37 °, and 45 °, under 12 h

darkness alternating with 12 h cool white fluorescent

light. Colour notations in parentheses are from Kor-

nerup & Wanscher (1984). Measurements were per-

formed in water and lactophenol. Photomicrographs

were obtained with a Leitz Dialux 20 EB microscope.

Scanning electron microscopy techniques were as

described by Figueras & Guarro (1988).

Mycol. Res. 106 (8) : 975–983 (August 2002). # The British Mycological Society

DOI: 10.1017}S0953756202006299 Printed in the United Kingdom.

New species of Thielavia, with a molecular study of

representative species of the genus

New species of Thielavia 976

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

Taxon Origin Strain no. EMBL no.

Gelasinospora bonaerensis soil ; Argentina FMR 5962 AJ002029

Melanocarpus thermophilus soil ; Irak FMR 6190 AJ271586

Thielavia appendiculata rabbit dung, UK CBS 731.68 AJ271584

T. arenaria (T) desert soil ; Egypt CBS 507.74 AJ271576

T. arxii sp. nov. soil ; Easter Island, Chile FMR 5875 AJ271580

T. australiensis (T) nesting material of incubator bird; Australia CBS 493.74 AJ271590

T. basicola on Arctostaphylos uva-ursi ; Switzerland CBS 229.82 AJ271591

T. coactilis salt marsh; Kuwait IMI 182707 AJ271585

T. fragilis (T) rhizosphere of Pennisetum thyphoideum ; India IMI 169641 AJ271578

T. hyalocarpa desert soil ; Egypt CBS 646.74 AJ271583

T. hyrcaniae Niger CBS 773.85 AJ271581

T. intermedia sp. nov. soil ; India FMR 5594 AJ271588

T. microspora saline desert soil ; Kuwait CBS 808.73 AJ271577

T. minuta cotton jacket ; Switzerland CBS 541.76 AJ271587

T. ovispora (T) root of Avena sativa ; Ukraine CBS 165.75 AJ271593

T. subthermophila on Vignia radiata ; India IMI 319643 AJ271575

T. terrestris ground soil ; IFO 9732 AJ271589

T. terricola on Polygala senega ; IFO 30068 AJ271579

T. minor on leaf of Elaeis guineensis ; CBS 611.74 AJ271582

T. tortuosa soil ; India FMR 5780 AJ271592

(T), ex-types ; CBS, Centraalbureau voor Schimmelcultures ; FMR, Facultad de Medicina de Reus; IFO, Institute for Fermentation, Osaka;

IMI, CABI Bioscience UK Centre; EMBL, European Molecular Biology Laboratory.

DNA extraction, amplification and sequencing

The strains sequenced are shown in the Table 1.

Gelasinospora bonaerensis (Stchigel, Cano & Guarro

1998) was used as outgroup. Fungal DNA was isolated

as described by Guillamo! n et al. (1996). Strains were

grown at 28 ° in Sabouraud broth (Difco) in Ehrlen-

meyer flasks shaken at 20 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

incubed for 1 h at 65 ° in 2 ml of extraction buffer

(80±2 m TrisHCI pH 8±0, 0±25 m NaCI, 25 m

EDTA, 0±5% SDS). The lysate was extracted with

phenol–chloroform–isoamyl alcohol solution (25:24:1,

by vol.) 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 TrisHCI pH 8±0, EDTA 1 m). The

rDNA ITS region containing ITS 1 and ITS 2 and the

intervening 5.8S rRNA gene were amplified as described

by Gene! et al. (1996), by using a Perkin Elmer 2400

thermal cycler (Perking Elmer Cetus, Emervyville, CA).

The primers ITS5 (5«-GGAAGTAAAAGTCGTAAC-

AAGG-3«) and ITS4 (5«-TCCTCCGCTTATTGATA-

TGC-3«) (White et al. 1990) were used. The amplifi-

cation 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 last 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 com-

parison 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 were run

on a 310 DNA sequencer (Applied Biosystems).

Nucleotide sequence alignment and analyses

The sequences compared in this paper were aligned

using CLUSTAL W, version 1.5, of multiple sequence

alignment computer program (Thompson, Higgins &

Gibson 1994). Cladistic analyses using the neighbour-

joining method (Saitou & Nei 1987) with confidence

values for individual branches determined by bootstrap

analyses (500 pseudoreplicates) was performed with the

MEGA 1.0 program (Kumar, Tamura & Nei 1993).

The nucleotide composition, the observed frequencies

from pairwise comparisons, and the alignment gap

sequences were performed with the MEGA 1.0 pro-

gram. The EMBL accession numbers of the sequences

are in Table 1.

RESULTS

Taxonomy

Thielavia intermedia Stchigel & Guarro, sp. nov.(Figs 1–8)

Mycelium ex hyphis hyalinis vel brunneis, ramosis, anasto-

mosans, septatis, 1–5 µm diam composito. Coloniae 22–25 °Cin agaro cum decocto tuberorum et carotarum crescentes

supra 70 mm diam post 14 d, brunneae, expansae, planae,

granulosae; reversum brunneum; sine exsudatum. Ascomata

superficialia vel immersa, globosa, non-ostiolata, translucida,

atrobrunnea vel nigrae, glabrae, 130–300 µm diam, peridium

7–8 stratiorum compositum, 10–15 µm crassum, ex textura

A. M. Stchigel and others 977

1

2

3

Figs 1–3. Thielavia intermedia (FMR 5594). Fig. 1. Detail of the peridium. Fig. 2. Asci with inmature ascospores.

Fig. 3. Ascospores. Bar¯ 10 µm.

epidermoidea vel textura intrincata compositum; paraphisibus

nullis. Asci octospori, sub-globosi vel late ellipsoidei, brevi-

stipitati, 30–40¬22–30 µm, tenuitunicati, evanescentibus.

Ascosporae obovatis vel limoniformis, brunneae, laevis,

12–17¬9–12 µm, cum poro germinalibus apicalis unicus,

protrudente. Status conidialis nullis.

Typus : India : Rajasthan State : Jaipur, 26° N, 75° E, isol.ex

soil, 29 Oct. 1995, J. Guarro [isol. A. M. Stchigel ] (IMI

370868 – holotypus, FMR 5594 – cultura ex-typo).

Mycelium composed of hyaline to brown, branched,

anastomosing, septate, smooth hyphae, 1–5 µm broad.

Colonies on PCA attaining over 70 mm diam in 14 d at

room temperature, brown (M 6E4), expanding, flat,

granulose due to ascomata production; the reverse

present the same color than the surface; exudate and

soluble pigment absent. Ascomata superficial to im-

mersed, globose, non-ostiolate, translucent, dark brown

to black, glabrous, 130–300 µm diam; peridium 7–8-

layered, 10–15 µm thick, textura epidermoidea to textura

intrincata, composed of thickened, brown to dark

brown, irregular cells, 4–15¬3–13 µm; paraphyses

absent. Asci 8-spored, subglobose to broadly ellipsoidal,

short-stipitate, 30–40¬22–30 µm, thin-walled, evan-

escent. Ascospores obovate to limoniform, hyaline when

young and dark brown when mature, one-celled,

smooth walled, 12–17¬9–12 µm, with one distinctive,

apical, protruding, 1 µm diam, germ pore. Anamorph

not observed.

Colonies on OMA growing at room temperature

show the same characteristics as those on PCA. Colonies

on MEA growing at room temperature, attaining a

diam over 70 mm in 14 d, raw umber (M 5F8),

expanding, flat to slightly cottony, granulose by

ascomata production; exudate hyaline; reverse raw

umber (M 5F8); soluble pigment orange yellow.

Colonies on PDA growing at room temperature,

attaining a diam over 70 mm in 14 d, yellowish white to

greyish yellow (M 4A2 to 4B3), cottony, zonate ;

exudate hyaline; reverse raw umber to deep yellow

(M 5F8 to 4A8); soluble pigment orange yellow. Asco-

mata sterile produced. Thermotholerant. At 37 ° grow-

ing rapidly, producing white colonies, ascomata absent.

At 45 ° and at 15 ° growing scarcely, producing white

colonies ; ascomata absent. No growth below 12 °.The main features of the T. intermedia ITS 1-2 and

5.8S rDNA region sequences are : 498 bp; 117 A;

152 C; 124 G and 105 T. The location of the ITS 1

region is from nucleotides 7 to 154, the gene 5.8S rRNA

from nucleotides 155 to 328, and the ITS 2 region from

nucleotides 329 to 465.

T. intermedia shows certain similarities with T. fragilis

and T. ovispora. All have obovate ascospores, but these

New species of Thielavia 978

4 5

6

87

Figs 4–8. Thielavia intermedia (FMR 5594). Fig. 4. Detail of the peridium. Fig. 5. Asci. Figs 6–7. Mature ascospores.

Fig. 8. Ascospores (SEM). Bar Figs 4–7¯ 15 µm, Fig. 8¯ 12±5 µm.

A. M. Stchigel and others 979

9

10

Figs 9, 10. Thielavia arxii (FMR 5875). Fig. 9. Detail of the peridium. Fig. 10. Ascospores. Bar¯ 10 µm.

are smaller in T. fragilis (11–14¬6–7 µm) and T. ovi-

spora (8–10¬5±5–6±5 µm).

Thielavia arxii Stchigel & Guarro, sp. nov. (Figs 9–16)

Mycelium ex hyphis sub-hyalinis vel brunneis, ramosis,

anastomosans, septatis, 2–5 µm diam composito. Coloniae

22–25 °C in PCA crescentes ad 35–45 mm diam post 14 d,

fusco-grisae, expansae, planae, granulosae; reversum fusco-

griseum; exsudatum hyalinum. Ascomata superficialia, glo-

bosa, non-ostiolata, translucida, brunnea, glabrae, 40–250 µm

diam; peridium 3–5-stratiorum compositum, 5–10 µm cras-

sum, ex textura epidermoidea compositum; paraphisibus

nullis. Asci octospori, sub-globosi vel late ellipsoidei, brevi-

stipitati, 35–55¬25–40 µm, tenuitunicati, evanescentibus.

Ascosporae late fusiformis, brunneae vel fusco-brunneae,

laevis, 12–22¬8–12 µm, cum poro germinalibus sub-apicalis

vel lateralis unicus. Status conidialis nullis.

Typus : Chile : Easter Island : Hanga-Roa, 27.1° S, 109.4° W,

from soil, 15 Sept. 1995, L. Zaror [isol. A. M. Stchigel ] (IMI

374725 – holotypus, FMR 5875 – cultura ex-typo).

Mycelium composed of subhyaline to brown, branched,

anastomosing, septate, smooth hyphae, 2–5 µm broad.

Colonies on PCA attaining a diam of 35–45 mm in 14 d

at room temperature, dark grey, expanding, flat,

granulose by ascomata production; exudate hyaline

and soluble pigment, when present, olive; reverse dark

grey. Ascomata superficial, globose, non ostiolate,

translucent, brown, glabrous, 40–250 µm diam; per-

idium 3–5-layered, 5–10 µm thick, textura epidermoidea,

composed of light brown, irregular cells, 3–18¬3–

13 µm; paraphyses absent. Asci 8-spored, subglobose

to broadly ellipsoidal, 35–55¬25–40 µm, thin-walled,

evanescent. Ascospores broadly fusiform, hyaline when

young and brown to dark brown when mature, one-

celled, smooth walled, 12–22¬8–12 µm, with one sub-

terminal to medial distinctive germ pore, 1–2 µm diam.

Anamorph absent.

Colonies on OMA growing at room temperature

similar to those shown on PCA, attaining a diam upon

70 mm in 14 d. Colonies on PDA growing at room

temperature, attaining a diam over 70 mm in 14 d,

white to dark grey, expanding, felty to cottony, radiate,

sulcate, and granulose by ascomata production; exudate

pale yellow; reverse olive brown to deep yellow (M 4F8

to M 4A6); soluble pigment orange yellow. Colonies on

MEA similar to those growing on PDA, but not sulcate

and slightly radiate. Thermotholerant. At 37 ° and at

42 °, growing rapidly, producing white colonies and

fertile ascomata; at 45 ° growing rapidly, without

production of ascomata. At 15 ° growing scarcely,

without production of ascomata. No growth below

12 °.The main features of the T. arxii ITS 1-2 and 5.8S

rDNA region sequences are : 529 bp; 117 A; 158 C;

139 G and 115 T. The location of the ITS 1 region is

from nucleotides 7 to 173, the gene 5.8S rRNA from

nucleotides 174 to 348, and the ITS 2 region from

nucleotides 349 to 493.

T. aegyptiaca, T. appendiculata, T. arenaria, T. co-

actilis, T. hyrcaniae and T. subthermophila, also have

ascospores with a subapical to lateral germ pore.

However, the ascospores of T. arxii are bigger than

New species of Thielavia 980

11 12

13

14

15 16

Figs 11–16. Thielavia arxii (FMR 5875). Fig. 11. Ascoma. Figs 12–15. Ascospores. Note the germ pore in lateral to medial

position. Fig. 16. Ascospores. Note the ascospore showing the germ pore (arrow) (SEM). Bar Figs 11–15¯ 10 µm,

Fig. 16¯ 10 µm.

A. M. Stchigel and others 981

94

99

T. arxii

T. terricola

T. hyrcaniae

T. minor

T. hyalocarpa

T. fragilis

T. coactilis

T. appendiculata

T. terrestris

T. basicola

T. australiensis

T. tortuosa

Melanocarpus thermophilus

T. minuta

T. microspora

T. subthermophila

T. arenaria

T. intermedia

Gelasinosporabonaerensis

0.01

86

100

94

67

92

57

92

62

Fig. 17. Neighbour-joining tree based on the nucleotide sequences of ITS – 5.8S rRNA regions. Branch lengths are

proportional to genetic distance. The scale bar represents 0±1% sequence divergence. Bootstrap replication frequences

greatest than 50% are indicated above the internodes.

those of T. aegyptiaca (13–14¬8–10 µm), T. arenaria

(9–12±5¬6–7±5 µm), T. coactilis (9–12±5¬(5–)6–7 µm)

and T. hyrcaniae (12–15¬5–6 µm). T. appendiculata

and T. subthermophila have ascospores of similar size to

those of the new species, but T. appendiculata has setose

ascomata and T. subthermophila has ascomata associ-

ated with numerous aleurioconidia which are absent in

T. arxii. Moreover, T. arxii is the only species of the

genus that has a germ pore in a median position in the

ascospores.

Molecular results

The phylogram obtained from the analysis of the ITS-

5.8S rDNA sequences using the neighbour-joining

method (Fig. 17) in general has clades with low

bootstrap values. Some of the terminal clades received

consistent statistical support which demonstrated some

species of the genus are genetically very close. The new

species Thielavia intermedia constituted a basal clade.

DISCUSSION

The main clade is formed by the species studied, apart

from one newly described (Thielavia intermedia), and

was divided into two sister clusters supported by a

bootstrap index of 57%. One grouped T. microspora,

T. subthermophila and T. arenaria, with a bootstrap

value of 86%; these species have ellipsoidal ascospores

and aleurioconidia. The terminal group constituted by

T. subthermophila and T. arenaria received a bootstrap

value of 100%; both species are thermotolerant and

have ascospores with a subapical to lateral germ pore,

and are differentiated only in their sizes. The second

New species of Thielavia 982

cluster was of the remaining species and comprised two

clades that received a low statistical support (48%).

The first clade is composed of T. tortuosa, Melanocarpus

thermophilus and T. minuta, grouped together with a

bootstrap index of 67%. Moreover, M. thermophilus

and T. minuta clustered with a very high bootstrap

value (94%); these species have striking morphological

similarities, short, septate and rugose setae, a peridium

of textura epidermoidea, and 8-spored, broadly ovoid

asci, differing only in temperature requirements and

morphology of the ascospores (Guarro et al. 1996).

Melanocarpus and Thielavia are distinguished by per-

idium type (textura angularis in Melanocarpus and

textura epidermoidea in Thielavia), the morphology of

the ascospores (oblate, thick-walled and opaque in

Melanocarpus and fusiform, ellipsoidal or ovate, thin-

walled and brownish in Thielavia), and in the ana-

morphs when present (Chrysonilia-like arthroconidia in

Melanocarpus, and Chrysosporium-like aleurioconidia

in Thielavia) (von Arx et al. 1988). However, it is

probable that future molecular studies will demonstrate

that these genera are not too genetically distinct. These

species are morphologically distant from T. tortuosa,

which has a peculiar combination of cylindrical, sinuose

asci and large ascospores with a subapical to lateral

germ pore.

The other clade was successively ramified forming

several branches also with a low confidence value.

However, three groups received strong statistical

support. One of them is composed of T. australiensis,

T. basicola and T. terrestris, with a bootstrap value of

92%. T. australiensis and T. terrestris have a thermo-

philic or thermotolerant ecology, and their ascospores

are morphologically similar. However, T. australiensis

has a Chrysosporium-like anamorph, and T. terrestris

produces enteroblastic conidia in chains. T. basicola is

not close morphologically to those, because it has

inequilateral ovate-fusiform ascospores and lacks an

anamorph. Two morphologically related species,

T. appendiculata and T. coactilis, were placed in a clade

supported by a bootstrap value of 62%. They show

ellipsoid to slightly fusiform ascospores with a subapical

germ pore. However, T. appendiculata can be differen-

tiated from T. coactilis by the presence of setose

ascomata, larger ascospores, and the absence of

chlamydospores. Finally, in another clade the new

species T. arxii was placed together with T. terricola,

T. hyrcaniae and T. minor, they formed a well defined

clade, supported by a bootstrap value of 99%. On the

basis of the morphology of T. terricola and T. minor

(ellipsoid ascospores with a terminal germ pore), we

expected them to cluster together, but not with the

other two species of the clade which show clearly

distinct features. T. arxii is easily distinguishable by its

fusiform ascospores with a lateral to median germ pore,

and is thermotolerant, while T. terricola and T. minor

have ellipsoidal ascospores with a terminal germ pore,

and are mesophilic.

This study confirmed that the ITS region is highly

conserved in Sordariales, as indicated previously (Stchi-

gel et al. 1998, 1999), and that it is not very useful to

establish phylogenetical relationships at species level.

We are currently evaluating the usefulness of the LSU

rDNA gene for this purpose.

ACKNOWLEDGEMENTS

We are indebted to Lori M. Carris (Washington State University) and

David H. Griffin (State University of New York) for useful editorial

suggestions on this manuscript, to Luı!s Zaror (Universidad de

Valdivia) for providing the Chilian soil samples, and to the curators

of CBS, IFO and IMI for kindly providing fungal cultures.

REFERENCES

Booth, C. (1961) Studies of pyrenomycetes : VI. Thielavia, with notes

on some allied genera. Mycological Papers 83 : 1–15.

Chen, K.-Y. & Chen, Z.-C. (1996) Thielavia pingtungia sp. nov., a

thermophilic ascomycete from Taiwan. Mycotaxon 60 : 241–247.

Figueras, M. J. & Guarro, J. (1988) A scanning electron microscopic

study of ascoma development in Chaetomium malaysiense. Mycolo-

gia 80 : 298–306.

Gene! , J., Guillamo! n, J. M., Guarro, J., Pujol, I. & Ulfig, K. (1996)

Molecular characterization, relatedness and antifungal suscep-

tibility of the basidiomycetous Hormographiella species and

Coprinus cinereus from clinical and environmental sources. Antonie

van Leewenhoek 70 : 49–57.

Guarro, J., Abdullah, S. K., AI-Bader, S. M., Figueras, M. J. &

Gene! , J. (1996) The genus Melanocarpus. Mycolological Research

100 : 75–78.

Guillamo! n, J. M., Cano, J., Ramo! n, D. &Guarro, J. (1996) Molecular

differentiation of Keratinomyces (Trichophyton) species. Antonie

van Leewenhoek 69 : 223–227.

Kornerup, A. & Wanscher, J. H. (1984) Methuen Handbook of

Colour. 3rd. edn. Eyre Methuen, London.

Kumar, S., Tamura, K. & Nei, M. (1993) MEGA: molecular

evolutionary genetics analysis. Version 1.0. Pennsylvania State

University, Philadelphia.

Ito, T., Okane, I. & Nakagiri, A. (1998) Thielavia aurantiaca, a new

species from Japanese soil. Mycoscience 39 : 93–96.

Malloch, D. & Cain, R. F. (1973) The genus Thielavia. Mycologia 65 :

1055–1077.

Mouchacca, J. (1973) Les Thielavia des sols arides : espe' cies nouvelles

et analyse ge!ne! rique. BulletıUn de la SocieU teU Mycologique de France

89 : 295–311.

Saitou, N. & Nei, M. (1987) The neighbour-joining method: a new

method for reconstructing phylogenetic trees. Molecular Biology

and Evolution 4 : 406–425.

Stchigel, A. M., Cano, J. & Guarro, J. (1998) A new species of

Gelasinospora from Argentina. Mycological Research 102 : 1405–

1408.

Stchigel, A. M., Sague! s, M., Cano, J. & Guarro, J. (1999) Three new

thermotolerant species of Corynascus (Sordariales, Chaetomiaceae)

from soil, with a key of the known species. Mycological Research

104 : 879–887.

Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) CLUSTAL

W: Improving the sensitivity of progressive multiple sequence

alignment through sequence weighting, positions-specific gap

penalties and weight matrix choice. Nucleic Acids Research 22 :

4673–4680.

von Arx, J. A. (1973) Ostiolate and non-ostiolate pyrenomycetes.

Proceeding of the Koninklijke Nederlandse Akademie van Weten-

schappen 76 : 289–296.

von Arx, J. A. (1975) On Thielavia and some similar genera of

ascomycetes. Studies in Mycology 8 : 1–29.

von Arx, J. A., Figueras, M. J. & Guarro, J. (1988) Sordariaceous

ascomycetes without ascospore ejaculation. Nova Hedwigia 94 :

1–104.

A. M. Stchigel and others 983

Warcup, J. H. & Baker, K. F. (1963) Ocurrence of dormant

ascospores in soil. Nature 197 : 1317–1318.

White, T. J., Bruns, T., Lee, S. & Taylor, J. (1990) Amplification and

direct sequencing of fungi ribosomal RNA genes for phylogenetics.

In PCR Protocols : a guide to methods and applications (M. A.

Innis, D. H. Gelfand, J. J. Sninsky & T. J. White, eds) : 315–322.

Academic Press, San Diego.

Corresponding Editor: D. L. Hawksworth