Molecular phylogeny of the mycorrhizal desert truffles ... - Mycologia

Mycologia, 94(2), 2002, pp. 247–259. 

2002 by The Mycological Society of America, Lawrence, KS 66044-8897 

Jesús Díez 1 

Molecular phylogeny of the mycorrhizal desert truffles (Terfezia and 

Tirmania), host specificity and edaphic tolerance 

UMR 1136 INRA-UHP ‘‘Interactions Arbres/ 

Micro-organismes’’, INRA-Nancy, F-54280 

Champenoux, France 

José Luis Manjón 

Dpto. de Biología Vegetal, Universidad de Alcalá, E- 

28871 Alcalá de Henares (Madrid), Spain 

Francis Martin 

UMR 1136 INRA-UHP ‘‘Interactions Arbres/ 

Micro-organismes’’, INRA -Nancy, F-54280 

Champenoux, France 

Abstract: Terfezia and Tirmania, so called desert 

truffles, are mycorrhizal fungi mostly endemic to arid 

and semi-arid areas of the Mediterranean Region, 

where they are associated with Helianthemum species. 

The aim of this work was to study the phylogenetic 

relationships in these pezizalean hypogeous fungi. 

The restriction fragment length polymorphism 

(RFLP) and DNA sequences of internal transcribed 

spacers (ITS) of the nuclear rDNA were studied for 

several morphological species, Terfezia arenaria, T. 

boudieri, T. claveryi, T. leptoderma, T. terfezioides 

(Mattirolomyces terfezioides), Tirmania nivea and T. 

pinoyi. The sequences were analyzed with distance 

and parsimony methods. Phylogenetic analyses indicated 

a close genetic relationship between Tirmania 

and Terfezia. They may have arisen from a single evolutionary 

lineage of pezizalean fungi that developed 

the hypogeous habit as an adaptation to heat and 

drought in Mediterranean ecosystems. This analysis 

also supports the re-establishment of the genus Mattirolomyces. 

The genera Tirmania and Terfezia were 

monophyletic, and morphological species corresponded 

to phylogenetic species. The Tirmania clade 

comprises desert truffles with smooth spores and amyloid 

asci, which were found in deserts. The Terfezia 

clade grouped species found in semi-arid habitats 

having ornamented and spherical spores. These species 

are adapted to exploit different types of soil (either 

acid or basic soils) in association with specific 

hosts (either basophilous or acidophilous species). 

Although other factors might also play a role, host 

specialization and edaphic tolerances (fungus and/ 

Accepted for publication August 7, 2001. 

1 Corresponding author, Email: diezmuriel@yahoo.com 

247 

or host tolerances) might be the key in the species 

diversity of these genera. 

Key Words: fungal evolution, Helianthemum, internal 

transcribed spacer, mycorrhizal fungi 

INTRODUCTION 

Pezizales are widespread Ascomycetes with either enclosed 

underground (hypogeous) or exposed (epigeous) 

fruit bodies (Trappe 1979). The hypogeous 

ascocarps of these fungi are known as truffles 

(Trappe 1992). In the Mediterranean region, hypogeous 

fungi colonize a variety of forests and semi-arid 

ecosystems. These fungi frequently establish mutualistic 

associations with vascular plants via specialized 

nutrient-gathering organs called mycorrhizas (Trappe 

1992). 

Many hypogeous fungi occurring in semi-arid ecosystems 

of the Mediterranean Basin belong to the 

genera Tirmania, Terfezia and Picoa (Alsheikh and 

Trappe 1983a, b, Moreno et al 1986, 1991, 2000, 

2001). Most of them are endemic to the Mediterranean 

region and establish mycorrhizal symbioses with 

members of the Cistaceae, mainly with Helianthemum 

species (FIG. 1) (Dexheimer et al 1985, Fortas and 

Chevalier 1992). These plants and their associated 

mycota may play a major role in the maintenance of 

Mediterranean shrublands and xerophytic grasslands, 

and thus in preventing erosion and desertification 

(Honrubia et al 1992). 

Morphological characters have been used to describe 

different species of desert truffles; i.e., spore 

and peridium morphology, gleba colour, odor and 

other organoleptic characters. These fungi, however, 

are difficult to identify at the species level. Regarding 

the systematics of desert truffles, the use of morphological 

features is problematic, because of the reduced 

set of morphological characters and their homoplasy. 

Ascocarp features are homoplastic as a result 

of parallel evolution of independent lineages of 

epigeous/hypogeous fruit bodies during the evolutionary 

history of the Pezizales (Trappe 1979). This 

accounts for the early artificial classifications of hypogeous 

fungi in the order Tuberales. Tuberales included 

several hypogeous families with similar morphology 

originating by parallel or convergent evolution 

(O’Donnell et al 1997). Realizing that the Tub-

248 MYCOLOGIA 

erales artificially grouped many Pezizales, Trappe 

(1979) transferred some of families from Tuberales 

to Pezizales and amended some families in Pezizales 

to include related hypogeous fungi. He also amended 

the Pezizaceae Fries sensu Korf to accommodate 

several related hypogeous taxa lacking forcible spore 

discharge, such as Tirmania. In addition, based on a 

comparative morphological study, Trappe (1979) included 

Terfezia in the Terfeziaceae and Picoa in the 

Balsamiaceae. 

The increasing amount of molecular phylogenetic 

data now available has led to a continuing revision of 

the hypogeous Ascomycetes (Gargas and Taylor 1995, 

Spatafora 1995, Landvik et al 1997, O’Donnell et al 

1997, Harrington et al 1999). O’Donnell et al (1997) 

provided support for the occurrence of independent 

lines of epigeous/hypogeous fruit body evolution in 

the Pezizales. This has affected classification of the 

desert truffles. Moreover, analysis of the 18S rDNA 

sequences has revealed a close relationship between 

Terfezia and the Pezizaceae (Percudani et al 1999, 

Norman and Egger 1999). Picoa is not closely related 

to the Pezizaceae (see O’Donnell et al 1997), where 

Picoa carthusiana Tul. & Tul. is referred to by the 

synonym Leucangium carthusianum (Tul.) Paol. 

Hence, it will be not studied in the present work. 

Subgeneric relationships within the Pezizales have 

been subjected to molecular analysis in few cases 

(Harrington and Potter 1997, Roux et al 1999, Norman 

and Egger 1996, 1999). Apart from studies of 

the genus Tuber (Roux et al 1999), no recent work 

has addressed subgeneric relationships within the hypogeous 

genera. In particular, no work has been published 

on the evolutionary history of desert truffles. 

The phylogenetic concept of species requires that 

species represent a monophyletic set of organisms. In 

the case of desert truffles, species delimitation by 

morphological characters seems to be consistent. 

However, molecular phylogenetic analyses are needed, 

in order to verify whether morphological species 

of desert truffles also represent phylogenetic species. 

The present work focuses on two major genera of 

pezizalean hypogeous fungi (Tirmania and Terfezia), 

and deals with the main morphological species that 

occur in the Mediterranean region. Because of the 

limited capacity of the 18S rDNA to resolve intrageneric 

relationships in hypogeous ascomycetes (Percudani 

et al 1999), we used the faster-evolving internal 

transcribed spacer (ITS) of the nuclear rDNA. 

Restriction fragment length polymorphism (RFLP) 

analysis was supplemented by sequence analysis. Phylogenic 

analysis, together with the morphological and 

ecological data, allowed us to propose an evolutionary 

history for these genera. 

MATERIALS AND METHODS 

Fungal collections. Most specimens studied in this work 

were collected by the authors. Several collections deposited 

at the Herbarium of Alcalá University (AH) were also studied. 

Specimens were harvested in xerophilous shrublands 

and grasslands dominated by Helianthemum spp. Host and 

soil data are mainly based on our observations and on notes 

accompanying herbarium specimens. Voucher specimens of 

new collections were deposited in AH (Alcalá University 

Herbarium, Spain). Specimen collections are listed in TA- 

BLE I. 

We studied a wide collection of desert truffles from the 

Iberian Peninsula (Spain and Portugal), Morocco, Tunisia, 

Algeria, Israel and Kuwait. Specimens were identified as: 

Tirmania nivea (Des. : Fr.) Trappe, T. pinoyi (Maire) Malençon, 

Terfezia arenaria (Moris) Trappe, T. boudieri Chatin, 

T. claveryi Chatin, or T. leptoderma (described in detail in 

Moreno et al 1986, 1999, 2000, 2001). Variability in spore 

ornamentation was the most important morphological feature 

for discriminating between these species. A comparative 

table of major morphological features is provided (TA- 

BLE II). 

Mycelial cultures. Isolates were obtained from ascomata on 

modified Melin-Norkrans (MMN) agar medium (Marx 

1969). All cultures were maintained on MMN agar medium 

at 25 C in the dark. Isolates were deposited at the Collection 

of Ectomycorrhizal Fungi of the Plant Biology Department 

of the University of Alcalá. 

DNA extraction and ITS amplification. DNA was extracted 

from specimens belonging to collections of desert truffles 

from the Iberian Peninsula, Morocco, Tunisia, Algeria, Israel, 

and Kuwait and two collections of Mattirolomyces terfezioides. 

Samples for DNA were excised either from the 

edge of a mycelial colony or from the inner part of the 

ascocarp to avoid contamination by other microorganisms. 

Mycelial samples were preserved at 20 C pending DNA 

extraction. Approximately 20–50 mg of tissue was used for 

each DNA extraction, performed using the cetyl-trimethylammonium 

bromide (CTAB) protocol (Gardes et al 1991). 

The ITS regions of nuclear rDNA were amplified with ITS1 

and ITS4 primers (White 1990) as described by Henrion et 

al (1992) on a GeneAmp 9600 PCR thermocycler (Perkin 

Elmer, Inc.). Controls with no DNA were included in every 

set of amplifications to test for DNA contamination in reagents 

and reaction buffers. 

RFLP analysis and DNA electrophoresis. Variation in ITS 

sequences was assessed among at least three specimens of 

each collection by restriction fragment length polymorphism 

(RFLP) with the following enzymes: AluI, Hinf I, 

HhaI, and RsaI. Six to 10 L of the amplified ITS was digested 

with 2.5 units of each enzyme for 1 h at 37 C, according 

to the manufacturer’s instructions (GIBCO-BRL, 

Pasley, UK). The amplification and digestion products were 

fractionated with 1.5% regular agarose gels or 6% polyacrylamide 

gels in a 1 Tris-borate-EDTA buffer. The gels were 

stained with ethidium bromide and photographed under 

ultraviolet light. 174 phage DNA digested with HaeIII 

was included in the gels as a molecular size marker.

Sequencing of the amplified ITS regions. ITS regions from 

18 specimens displaying different RFLP patterns were sequenced 

(TABLE III). Amplified DNA was purified with 

GeneClean Kit (Bio101, Carlsbad, California, USA). DNA 

concentrations were quantified on agarose gels with a Low 

DNA Mass ladder (GIBCO-BRL, Pasley, UK) and sizes calculated 

with a 100-bp ladder (GIBCO-BRL). Sequencing reactions 

were performed directly on purified PCR products 

with ITS1 or ITS4 primers (White et al 1990). Both strands 

were sequenced with the Taq DyeDeoxy Terminator Cycle 

Sequencing Kit (Perkin Elmer, Norwalk, Connecticut, 

USA). The sequence products were analyzed with an ABI 

model 373 DNA fluorescent sequencer (Perkin Elmer). The 

sequences obtained were deposited in the National Center 

for Biotechnology Information (NCBI) GenBank (TABLE 

III). 

Sequence alignment and analysis. The search for sequence 

identity in the GenBank DNA database was conducted by 

Gapped BLAST (NCBI) (Altschul et al 1997). Three ITS 

sequences of T. boudieri were retrieved from GenBank and 

added to the alignment data set. The ITS sequences of the 

closely related species of Peziza (Norman and Egger 1999) 

were also retrieved from GenBank in order to verify whether 

the Tirmania/Terfezia clade is monophyletic after introduction 

of Peziza species. Sequences were aligned with 

MultAlin (Corpet 1988) at the ProDom web site (http:// 

www.toulouse.inra.fr/multalin.html) (INRA-CNRS, Toulouse). 

The sequence alignment was hand-edited and deposited 

in TreeBASE (accession number SN908) (http:// 

www.herbaria.harvard.edu/treebase/). 

Aligned sequences were analyzed by means of distance 

and parsimony methods. Tuber melanosporum was used as 

outgroup (GenBank accession no. U89359). Distances were 

calculated according to the Junkes and Cantor model, as 

D AB (3/4)ln[1 4/3(Su/I Su)][1 G/T] G/T. 

D AB is the distance between sequences A and B, I the number 

of identical nucleotides, Su the number of positions 

showing a substitution, G the number of gaps in one sequence 

with respect to the other, and T the sum of I, S and 

G. Insertions and deletions were taken into account. Tree 

topology was inferred by the Neighbor-Joining (NJ) method 

(Saitou and Nei 1987). The Bootstrap method (Felsenstein 

1985) was performed with 1000 replications to evaluate the 

reliability of tree topologies. Distance analysis and tree 

drawing were carried out with TreeCon (Van de Peer and 

De Wachter 1993). 

Maximum parsimony (MP) trees were inferred with the 

heuristic method with the aid of PAUP 3.1.1 (Swofford 

1993). Validity of the clades was tested by bootstrap analysis 

(Felsenstein 1985). The analysis was conducted with 1000 

bootstrap replications, retaining groups compatible with the 

50% majority-rule in the bootstrap consensus tree, with the 

Nearest-Neighbor Interchange (NNI) branch-swapping option, 

and no more than 600 trees (700 length) saved each 

replication. The 50% majority rule consensus tree is available 

on line at TreeBASE (SN908). Parsimony trees were 

drawn with the aid of TreeView (Page 1996). 

DíEZ ET AL: DESERT TRUFFLES PHYLOGENY 

RESULTS 

249 

RFLP profiles of amplified ITS. The ITS region was 

successfully amplified for 34 collections. In contrast, 

four old herbarium collections (nos. 6, 21, 26, and 

27) did not amplify, probably as a result of DNA degradation. 

Each species produced a characteristic 

RFLP pattern (A to G, TABLE IV), except for Terfezia 

arenaria and T. leptoderma. Terfezia arenaria was polymorphic 

for AluI and produced two RFLP profiles. 

Terfezia leptoderma was polymorphic for Hinf I and 

RsaI. Three sequences of T. boudieri retrieved from 

GenBank (AF092096–AF092098; strain type-1, type-2 

and type-3; Western Negev, Israel) gave predicted 

RFLP patterns for this species. 

Sequence comparisons. Search for similar sequences 

in the GenBank DNA database produced significant 

alignments with the ITS sequences of Terfezia boudieri 

(AF092096–AF092098) and Mattirolomyces terfezioides 

(AJ272442–AJ272445). The Genbank sequences of 

M. terfezioides showed 99% similarity with the sequence 

AF276681 of Mattirolomyces terfezioides (collection 

38, present study). Surprisingly, a search for 

sequence similarity using the BLASTn algorithm did 

not produce significant alignments with the Peziza/ 

Plicaria sequences reported by Norman and Egger 

(1999). In addition, the ITS sequences of P. badia 

and P. griseo-rosea (U40475 and U4047) were shorter 

and very different from those of Terfezia and Tirmania. 

This finding was unexpected, because they have 

been suggested to be closely related species to Terfezia 

arenaria (Norman and Egger 1999). Furthermore, 

no unambiguous alignment was achieved after 

the addition of these sequences of Peziza to the set 

of Terfezia and Tirmania ITS sequences. 

The variations in the length of the ITS sequences 

were often attributable to deletions and insertions. 

Gaps were therefore introduced in order to align the 

sequences. The total length of the alignment was 660 

positions. They comprised a small portion of the 

flanking 18S and 28S rDNA genes (11 and 36 bp 

respectively), the ITS1 region (nucleotides 12–247), 

the 5.8S rDNA (nucleotides 248–403), and the ITS2 

sequence (nucleotides 404–624). Sequence variability 

was most prominent within the ITS regions, which 

had several indels (e.g., all Terfezia and Tirmania species 

contained a 6-bp deletion between the nucleotide 

positions 33 and 39). Other deletions were specific 

to a set of species or just one species; e.g., the 

ITS1 sequence of Terfezia arenaria had a large deletion 

between positions 101–114 in the consensus. 

Phylogenetic inferred trees. The phylogenetic trees inferred 

by both distance-based (FIG. 2) and cladistic 

methods (FIG. 3) showed the same topology, in spite

250 MYCOLOGIA 

TABLE I. Collections of desert truffles used in the present work 

Collection 

no. a Origin Habitat b Host 

Spore 

morphology Phenetic species 

1* Kuwait Basic soil, AR Helianthemum salici- Smooth Tirmania nivea 

folium (L.) Mill. 

Chatin 

2** Spain, the Tabernas Sandy basic soil, 

— Smooth T. nivea (Des. : Fr) 

Desert, Almeria AR 

Trappe 

3** Tunisia, market of 

Tunis 

— — Smooth T. nivea 

4* Tunisia, market of 

Tunis 

— — Smooth T. nivea 

5* Algeria Acid soil, AR H. guttatum (L.) Mill. Subsmooth T. pinoyi (Maire) 

Malençon 

6** Morocco Acid soil, AR H. guttatum Subsmooth T. pinoyi 

7* Spain, Navalmoral, Sandy acid soils, H. guttatum Warty Terfezia arenaria 

Cáceres 

SAR 

(Moris) Trappe 

8* Spain, Navalmoral, Sandy acid soils, H. guttatum Warty Terfezia arenaria 

Cáceres 

SAR 


9** Spain, Navalmoral, Sandy acid soils, H. guttatum Warty Terfezia arenaria 

Cáceres 

SAR 


10* Spain, Campo Ar- Sandy acid soil, H. guttatum Warty T. arenaria 

añuelo, Cáceres SAR 

11** Spain, Campo Ar- Sandy acid soil, H. guttatum Warty T. arenaria 


12* Spain, Albuquerque, 

Cáceres 

Acid soil, SAR H. guttatum Warty T. arenaria 

13* Morocco, Mamora Acid soil, SAR H. macrosepalum (L.) 

Mill. 

Warty T. arenaria 

14* Morocco, Mamora, 

bought in a local 

market 

— — Warty T. arenaria 

15** Morocco, Mamora, 

bought in a local 

market 

— — Warty T. arenaria 

16* Portugal Acid soils, SAR H. guttatum Warty T. arenaria 

17** Algeria Basic soil, SAR H. salicifolium Reticulate 

and warty 

T. boudieri Chatin 

18* Kuwait Basic soil, SAR H. salicifolium Reticulate 

and warty 

T. boudieri 

19* Spain, Fuentidueña, Gypsiferous H. squamatum (L.) Reticulate T. boudieri 

Madrid 

shrubland, 

SAR 

Dum. Cours. 

and warty 

20** Spain, Valdeguerra, Calcareous soil, H. salicifolium Reticulate T. boudieri 

Madrid 

SAR 

and Warty 

21* Spain, Cabezamesa- Basic soil, SAR H. ledifolium and H. Reticulate T. boudieri 

da, Toledo 

salicifolium 

and warty 

22** Spain, The Toledo Basic soil, SAR H. salicifolium Reticulate T. boudieri 

Mountains 

and warty 

23* Morocco, market of 

— — Reticulate T. boudieri 

Tangier 

and warty 

24** Morocco, market of 

— — Reticulate T. boudieri 

Casablanca 

and warty 

25** Spain, Torres de la Calcareous soils Helianthemum spp. Reticulate T. claveryi Chatin 

Alameda, Madrid SAR 

basphilous 

26** Spain, Guadix, Gra- Basic soil, SAR Helianthemum spp. Reticulate T. claveryi 

nada 

basophilous 

27** Spain, Llanos de Basic soil, SAR Helianthemum spp. Reticulate T. claveryi 

Armilla, Granada 

basophilous

TABLE I. Continued 


Collection 

no. a Origin Habitat b Host 

Spore 

morphology Phenetic species 

28* Spain, Navalmoral, Sandy acid soil, H. guttatum Spiny T. leptoderma Tul. & 

Cáceres 

SAR 

C. Tul. 

29* Spain, Navalmoral, Sandy acid soil, H. guttatum Spiny T. leptoderma 

Cáceres 

SAR 

30** Spain, Campo Ar- Sandy acid soil, H. guttatum Spiny T. leptoderma 


31** Spain, Campo Ar- Sandy acid soil, H. guttatum Spiny T. leptoderma 


32** Spain, Cáceres Sandy acid soil, 

SAR 

H. guttatum Spiny T. leptoderma 

33** Spain, Trujillo, Cá- Sandy acid soil, H. guttatum Spiny T. leptoderma 

ceres 

SAR 

34** Spain, Cañaveral de Clayey slate de- Cistus ladanifer L. Spiny T. leptoderma 

León, Huelva rived soil, SAR 

35** Spain, Valencia Siliceous soil, 

SAR 

Pinus halepensis Mill. Spinyc T. leptoderma 

36** France, Auge-Fourvieille 

Clayey soil, SAR Quercus ilex L. Spinyc T. leptoderma 

37** Hungary, Csomad Forest plantation Robinia pseudoacacia Reticulate Mattirolomyces terfezioides 

(Matt.) Fischer 

38** Hungary, Surany Garden Ribes rubrum Reticulate M. terfezioides 

a DNA was extracted from mycelium (*) or fruit body (**). 

b Habitat: AR arid regions; SAR semi-arid regions. 

c Slightly smaller spore having shorter spines. 

TABLE II. Pezizalean desert truffles that naturally occur in the Mediterranean Region. Diagnostic morphological features, 

hosts and soil types 

Morphological 

species 

Main ascocarp 

features 

T. nivea Ascocarp off-white 

when young 

T. pinoyi Ascocarp white-yellowish 

when young 

T. arenaria Gleba with pinkish 

tones at maturity 

T. boudieri Thick peridium gleba 

red tones at maturity 

T. claveryi Gleba with red tones 

at maturity 

T. leptoderma Thin peridium, gleba 

with olivaceous 

tones at maturity 

Melzer’s reaction 

of asci 

Spore ornamentation 

and size 

Amyloid Smooth oval to ellipsoidal, 

16–18 12–14 

m 

Amyloid Subsmooth (minute 

warts) spherical, 15– 

20 m 

Non amyloid Warty spherical, 20–26 

m 

Non amyloid Reticulate-warty spherical, 

18–22 m 

Non amyloid Reticulate spherical, 18– 

22 m 

Non amyloid Spiny spherical, 17–25 

m a 

a Inmature ascocarps present slightly smaller spores (15–22 m) and shorter spines. 

b Habitat: AR arid regions, SAR semi-arid regions. 

Soil type, habitat b 

and host 

251 

Basic soils, AR, basophilousHelian- 

themum spp. 

Acid soils, AR H. 

guttatum 

Acid soils, SAR H. 

guttatum 

Basic soils, SAR basophilus 

spp. of 

Helianthemum 

Basic soils, SAR acidophilous 

spp. of 

Helianthemum 

Acid soils, SAR H. 

guttatum and Cistus 

ladanifer, 

Quercus ilex, Pinus 

halepensis

252 MYCOLOGIA 

TABLE III. Specimens used for sequencing 

Phenetic species a 

Tirmania nivea (03) 

T. pinoyi 

Terfezia claveryi 

(02) 

(04) 

(01) 

(05) 

(25) 

(26) 

T. boudieri (18) 

(17) 

T. arenaria (10) 

(11) 

T. leptoderma (30) 

(31) 

(34) 

(35) 

(36) 

Mattirolomyces terfezioides (37) 

(38) 

ITS-RFLP 

patterns 

A 

A 

A 

A 

B 

C 

C 

D 

D 

E1 

E2 

F1 

F1 

F2 

F3 

F4 

G 

G 

Sample 

no. b Origin Soil type Host 

tun08* 

tab04** 

tun06* 

niv05* 

pin01* 

cly06** 

alc03** 

bou04* 

mot08** 

are19* 

are20** 

lpt12* 

lpt08** 

jar02** 

val06** 

enc07** 

rob01** 

rib02** 

Tunisia 

Spain 

Tunisia 

Kuwait 

Algeria 

Morocco 

Spain 

Kuwait 

Algeria 

Spain 

Spain 

Spain 

Spain 

Spain 

Spain 

France 

Hungary 

Hungary 

— 

Basic 

— 

— 

— 

Basic 

Basic 

Basic 

Basic 

Acid 

a In brackets collection no. according to TABLE I. 

b The ITS regions were amplified from mycelium (*) or fruit bodies (**). 

of slight differences in branch stability among equivalent 

branches. Trees branched into two main clades, 

which were well supported by bootstrap values: the 

Mattirolomyces clade (100% NJ and MP) and the Tirmania/Terfezia 

clade (99% NJ and MP). Terminal 

clades of the inferred trees corresponded exactly to 

morphological species (FIG. 2) and were related to 

ecological features and hosts (FIG. 3). 

The Tirmania clade comprised desert truffles with 

smooth spores and amyloid asci, which were found 

in deserts. The Tirmania nivea terminal clade (100% 

NJ and MP) was composed of collections with oval to 

ellipsoidal spores, found under basophilous species 

of Helianthemum (e.g., H. salicifolium). Tirmania pinoyi, 

which has spherical spores and occurs under the 

acidophilous plant H. guttatum, was a sister species 

of T. nivea. 

The Terfezia clade (97% NJ, 85% MP) grouped species 

found in semi-arid habitats and with ornamented 

and spherical spores. The first subclade comprises 

well-supported terminal clades corresponding to Terfezia 

spp. However, relationships between them were 

not well resolved. Terfezia arenaria, which displays 

warty spores and occurs in sandy acid soils with H. 

guttatum (FIG. 1), was monophyletic and the corresponding 

terminal clade was well supported (100% 

NJ and MP). Terfezia boudieri and T. claveryi, which 

occur in basic soils under basophilous Helianthemum 

and possess reticulated and warty-reticulated spores, 

respectively, were similarly monophyletic (100% NJ 

Acid 

Acid 

Acid 

Acid 

Basic 

Basic 

— 

— 

Helianthemum salicifolium 

— 

— 

— 

H. guttatum 

H. ledifolium 

H. salicifolium 

H. salicifolium 

— 

H. guttatum 

H. guttatum 

H. guttatum 

H. guttatum 

Cistus ladanifer 

Pinus halepensis 

Quercus ilex 

Robinia 

— 

Genbank 

acc. no. 

AF276665 

AF276666 

AF276667 

AF276668 

AF276669 

AF276670 

AF276671 

AF276672 

AF276673 

AF276674 

AF276675 

AF276678 

AF276679 

AF396862 

AF396863 

AF396863 

AF276680 

AF276681 

and MP). The second subclade includes specimens 

with spherical spiny spores belonging to T. leptoderma 

(89% NJ, 95% MP). Terfezia leptoderma clade comprised 

specimens associated with several different 

hosts (Helianthemum guttatum, Cistus ladanider, 

Quercus ilex and Pinus halepensis), and were collected 

in different type of soils (either basic or acid soils). 

Thus, the molecular phylogenetic analysis indicated 

that morphological species represent phylogenetic 

species. 

DISCUSSION 

Previous studies have noted that desert truffles are 

sometimes difficult to distinguish on the basis of their 

morphology (Moreno et al 2000, 2001). In the present 

work, the RFLP profiles of the nuclear rDNA ITS 

sequences were consistent with species delimitation 

based on known morphological characters. In addition, 

ITS sequences were nearby homogenous within 

species (e.g., 0.3% nucleotide divergence within Terfezia 

arenaria) to polymorphic (up to 7% within T. 

leptoderma), and clearly polymorphic at the interspecific 

level (8%). 

The general morphological characteristics of Tirmania 

and Terfezia are described in Trappe (1979), 

and Alsheikh and Trappe (1983a) authored a monograph 

of Tirmania. Both genera are characterized by 

globose to turbinate ascocarps, with solid glebae 

formed of fertile pockets separated by pale sterile tra-

TABLE IV. Restriction size polymorphism in desert truffles (Tirmania and Terfezia spp.) and Mattirolomyces terfezioides. Size of the uncut ITS and restriction fragments 

in base pairs (bp) 

RFLP 

patterns 

Size of RFLP fragments 

AluI HhaI HinfI b RsaI 

Uncut 

ITS 

Collection nos. a Phenetic species 

1, 2, 3, 4 Tirmania nivea 620 390, 230 350, 270 210, 170, 120, 100 620 type A 

5, 6 T. pinoyi 630 380, 250 340, 290 200, 140, 130, 100, 40 480, 150 type B 

24, 25, 26, 27 Terfezia claveryi 630 380, 250 340, 290 300, 310 490, 90, 50 type C 

17, 18, 19, 20, 21, 22, 

23 T. boudieri 640 390, 250 350, 290 310, 230, 80 500, 90, 50 type D 

7, 8, 10, 11, 12, 13 T. arenaria (are19) 640 370, 270 330, 310 300, 320 490, 90, 60 type E1 

9, 11, 12, 13, 14, 15, 16 (are20) 640 640 330, 310 300, 320 490, 90, 60 type E2 

28, 29, 30, 31, 32, 33 T. leptoderma (lpt08) (lpt12) 620 390, 230 350, 270 310, 290 530, 90 type F1 

34 (jar02) 630 380, 250 340, 290 300, 310 400, 140, 90 type F2 

35 (val06) 640 390, 250 350, 290 310, 310 400, 140, 90 type F3 

36 (enc07) 620 380, 240 340, 280 300, 190, 110 530, 90 type F4 

37, 38 Mattirolomyces terfezioides 670 390, 280 370, 300 330, 140, 110, 80 580, 90 type G 


a See Table I for specimen data. 

b This enzyme also produced one or two 20 bp fragments. 

253 

mal veins. They have saccate to globose asci with up 

to eight clustered spores. Based on these morphological 

data, similar habitat and associated hosts, they 

have been considered as closely related genera. Our 

molecular analyses confirmed their close relationship. 

Moreover, the phylogenetic analyses indicate 

that both genera are monophyletic. Such a monophyly 

is in agreement with morphological data. Terfezia 

has non-amyloid asci and ornamented spores. In 

contrast, Tirmania has amyloid asci and smooth 

spores, both of which are features regarded as diagnostic 

characters at different taxonomic ranks. 

Trappe (1971) used Melzer’s reagent to distinguish 

Tirmania from Terfezia. Later, Trappe (1979) used 

this character to transfer Tirmania from the Terfeziaceae 

(non-amyloid asci) to the Pezizaceae (amyloid 

asci). Amyloid reaction seems to be diagnostic at the 

genus level. However, the strong statistical support 

for the Terfezia/Tirmania molecular clade (present 

work), together with the lack of amyloid asci in Matteriolomyces, 

suggest that this biochemical reaction is 

of limited value as diagnostic character at the family 

level. 

To reconstruct the molecular phylogeny of these 

fungi, we included the related hypogeous fungus 

Mattirolomyces terfezioides Fischer (Percudani et al 

1999). This fungus does not occur in the same habitats 

as Terfezia and Tirmania, but in temperate forests. 

This species seems to be specifically associated 

with Robinia pseudoacacia (Montecchi and Lazzari 

1993, Bratek et al 1996). It was probably co-introduced 

to Europe with Robinia pseudoacacia. The collection 

of M. terfezioides under Ribes rubrum in a garden 

in Hungary (collection no. 38, TABLE I) does not 

provide reliable information on its mycorrhizal status, 

because Robinia pseudoacacia is a common garden 

tree in Hungary. Mattirolomyces terfezioides was 

described by Fischer (1938) and then assigned to the 

genus Terfezia by Trappe (1971). Later, based on the 

analysis of the 18S rDNA sequence, Percudani (1999) 

suggested the re-establishment of the genus Mattirolomyces. 

The lack of nesting of Mattirolomyces terfezioides 

in the Terfezia clade (FIGS. 2 and 3) and the 

study of Norman and Egger (1999) support this reestablishment. 

According to molecular data (O’Donnell et al 

1997, Norman and Egger 1999, Percudani et al 

1999), Terfezia and other pezizalean ascomycetes 

such as Pachyphloeus, Mattirolomyces, and Cazia, may 

have evolved from ancestral epigeous pezizas towards 

a hypogeous habit. Some species of Pachyphloeus and 

Mattirolomyces combine a hypogeous ascoma with biseriate 

spores. This combination might represent an 

intermediate step between the uniseriate asci of epigeous 

Pezizaceae and globose asci of the genera Ter-

254 MYCOLOGIA 

FIG. 1. A common desert truffle in sandy acid soils of the semiarid regions in the Iberian Peninsula (Terfezia arenaria) 

and its mycorrhizal host plant Helianthemum guttatum. 

fezia and Tirmania. Analysis of the ITS sequences 

showed the separation of the Mattirolomyces from the 

Terfezia-Tirmania clade. They seem to represent two 

different evolutionary pezizalean lineages. This hypothesis 

is in agreement with the analyses of the 18S 

rDNA sequences by Norman and Egger (1999) and 

Percudani et al (1999), who found that Terfezia arenaria 

and Mattirolomyces terfezioides are not sister 

genera. Indeed, Pachyphloeus and Mattirolomyces species 

occur in temperate forests with cold winters and 

are associated with North Temperate trees (Trappe 

1979). In addition, M. terfezioides and Pachyphloeus 

melanoxanthus were sister species in Percudani 

(1999). They perhaps evolved hypogeous ascocarps 

to protect fruitbody development from frost, and rely 

on animals for their spore dispersal. This might be 

the case with most common forest-dwelling truffles 

fruiting in winter in Central Europe (e.g., Tuber melanosporum). 

In contrast, Terfezia and Tirmania occur 

in arid and semi-arid ecosystems in the Mediterranean 

region, associated with dwarf shrubs and herbaceous 

plants (Helianthemum spp.). The desert truffles 

fruit are vernal fungi. The Terfezia-Tirmana evolutionary 

lineages have probably evolved towards hypogeous 

fruitbodies as protection from the heat and 

drought of late spring in semi-arid and arid habitats. 

The evolution of epigeous fruitbodies towards hypogeous 

habits seems to have happened in several lineages 

of ectomycorrhizal fungi; selection for reduction 

of water loss has been proposed to explain the 

accelerated evolution of suilloid basidiocaps towards 

false truffles (e.g., Rhizopogon) through secotioid 

forms (Bruns et al 1989). 

Relationships between Peziza/Plicaria and Terfezia


FIG. 2. Neighbor-joining tree of 30 ITS/5.8S rDNA sequences of pezizalean truffles constructed with the Jukes and 

Cantor’s one-parameter distance method. Numbers in branches are the bootstrap values as percentage bootstrap replication 

from a 1000 replicate analysis. Scale represents the distance between isolates. Outlines delimit clusters, which correspond to 

morphological species. Square brackets delimit genera. Major diagnostic morphological characters are indicated. 

255

256 MYCOLOGIA 

FIG. 3. Rooted 50% majority rule consensus tree resulting from 1000 bootstrap replications of the parsimony analysis of 

the ITS/5.8 rDNA sequences (607 steps, consistency index, CI 0.81; retention index, RI 0.86; rescaled consistency index, 

RC 0.70; homoplasy index, HI 0.19). Analysis was conducted using the heuristic search algorithm of PAUP 3.1.1. 

(Swofford 1993). Numbers on the branches are the bootstrap values (%). Scale represents steps. Outlines indicate the 

different clades observed, which coincide with the different habitats (e.g., temperate forests, deserts and semi-arid areas). 

Habitat, soil types and hosts are indicated.

have been recently studied by 18S rDNA sequence 

analysis (Norman and Egger 1999, Percudani et al 

1999). Whereas Percudani et al (1999) indicated that 

P. badia is not a sister species of Terfezia, Norman and 

Egger (1999) reported Peziza badia and P. griseo-rosea 

as closely related to Terfezia. In the present study, Terfezia, 

Matteriolomyces, and Tirmania ITS did not produce 

significant sequence similarities with ITS of Peziza 

deposited in GenBank. This result is in agreement 

with Percudani et al (1999). The ITS sequences 

of P. badia and P. griseo-rosea (U40475 and U4047) 

are shorter and very different from those of Terfezia 

and Tirmania. In the present study, we wanted to verify 

whether the Tirmania/Terfezia clade was still 

monophyletic after introduction of Peziza species in 

the phylogenetic analyses. The lack of satisfactory 

alignments among ITS sequences of Tirmania/Terfezia 

and Peziza supports the divergence of these lineages 

and the monophyly of the Terfezia/Tirmania 

clade. 

The Tirmania-Terfezia lineage presented two wellsupported 

branching clades, one with smooth spores 

and amyloid asci (Tirmania spp.) and the other with 

ornamented spores and nonamyloid asci (Terfezia 

spp.). Species with smooth spores occur in desert areas 

of the Mediterranean region (Alsheikh and 

Trappe 1983b). In the Western Mediterranean Basin, 

its northernmost populations seem to be in the Tabernas 

Desert in Southern Spain (Moreno et al 

2000). In contrast, Terfezia species (ornamented 

spores) occur in semiarid areas and thus have a more 

northerly distribution; i.e., they are well represented 

all over the Mediterranean area of the Iberian Peninsula 

(Alvarez et al 1993, Moreno et al 2001). Although 

regarded as hypogeous fungi, the desert truffles 

eventually emerge above ground, resulting in a 

semi-hypogeous habit. Furthermore, it has been 

claimed that their ascocarps then dry in situ from the 

heat and drought of late spring (Trappe 1992). Trappe 

(1992) further suggested that in Tirmania the peridium 

collapses when dried and spores are then disseminated 

by dry winds. We have observed fruitbodies 

of Terfezia bitten by rodents, suggesting a potential 

role of animals in spore dissemination. 

Because today’s species of desert truffles seem to 

represent different species adapted to different types 

of soils, differences in the edaphic tolerance may account 

for species diversity. Terfezia boudieri and T. 

claveryi occur in marl-gypsum soils. In contrast, T. arenaria 

lives in siliceous sands. Terfezia leptoderma was 

found in association with Helianthemun guttatum in 

acid soil and with Cistus ladanifer in slate-derived 

soils. This fungus was also associated with Quercus ilex 

and Pinus halepensis in basic soils. We observed some 

differences in the spore morphology of the T. lepto- 


257 

derma. The small spores of specimens collected under 

pine and Q. ilex would fit those of T. olbiensis. 

Terfezia olbiensis was described with similar morphology 

to T. leptoderma, except for slightly smaller spores 

and shorter spines. However, there is a certain consensus 

that T. olbiensis is an immature form and a 

synonym of T. leptoderma (Moreno et al 1986, 2001, 

Alvarez et al 1993). The morphological species T. leptoderma 

might be either a species with wide edaphic 

tolerance and host range or a species complex; isolates 

occurring in Cistus scrubs and pine and Quercus 

sclerophilous woodlands could belong to distinct species 

with different host or/and edaphic specialization. 

Our sampling in these ecosystems is scanty and 

further study is necessary. 

In the Mediterranean region, other mycorrhizal 

taxa seem to present different edaphic tolerances 

and host ranges, i.e., the Pisolithus species complex 

(Díez et al 2000, 2001). As in the case of Terfezia 

species, ITS analyses suggested the presence of several 

Pisolithus species occurring in different soil types 

(basic, acid, and clayey slate-derived soils) and with 

specificity for particular indigenous hosts. 

In the case of Tirmania spp., Tirmania nivea collections 

analyzed in the present work were found in 

basic soils (e.g., collection no. 2) and associated with 

the basophilous plant H. salicifolium (e.g., no. 1). In 

contrast, T. pinoyi specimens were collected under 

the acidophilous plant H. guttatum (e.g., collections 

nos. 5–6). Larger surveys are needed to confirm 

whether T. nivea and T. pinoyi also have different 

edaphic tolerances and/or host adaptations. 

The distribution pattern of the desert truffles species 

seems to correlate so strongly with host, that the 

two factors (host specialization and soil pH) might 

have played a key role in their speciation. Furthermore, 

most species of Helianthemum forming mycorrhizas 

with desert truffles in the Mediterranean region 

show different edaphic tolerances. Whereas H. 

salicifolium and H. ledifolium occur in basic soils, other 

species of Helianthemum occur only in acid soils, 

e.g., H. guttatum. Soil features therefore have an impact 

on the distribution of the host plants. Distribution 

of desert truffles species according to soil features 

might therefore just reflect the edaphic tolerance 

of their hosts. 

The present phylogenetic analyses confirm that 

these morphological species are also phylogenetic 

species. Distance analysis indicated that they are well 

separated. Distance values within and among species 

are low, suggesting that these species diverged a short 

time ago. Estimation of reliable divergence-time rates 

for ITS sequences from mycorrhizal fungi would be 

invaluable in dating this divergence. A larger survey 

using multilocus molecular analyses is needed to de-

258 MYCOLOGIA 

termine the genetic structure of Terfezia and Tirmania 

populations. 

ACKNOWLEDGMENTS 

We thank Dr. James M. Trappe (Forest Service, Corvallis, 

Oregon), Dr. Thomas D. Bruns (University of California, 

Berkeley, California), and anonymous reviewers for critical 

comments on the manuscript. We are grateful to Dr. G. 

Chevalier and C. Dupré (INRA, Clermont-Ferrand, 

France), L. Khabar (University of Rabat, Morocco) and 

Romero de la Osa (Forest Service, Junta de Andalucía, 

Spain) for providing several strains and specimens. We also 

thank Dr. E. Jakucs (Eotvos Lorand University, Budapest, 

Hungary) for his comments on Mattirolomyces terfezioides, 

and Dr. G. Moreno (Alcalá University) for assistance in the 

taxonomic identification. We are also grateful to INIA (project 

SC98–030) and ‘‘Vicerrectorado de Investigación de la 

Univ. de Alcalá’’ (EO28/98) for their financial support. 

This work was supported by a postdoctoral fellowship (EU, 

Contract HPMF-CT-1999-00174) and a ‘‘Ramón y Cajal’’ 

contract from the MCyT (Spain) to J. Díez. 

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