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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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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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/or host tolerances) might be the key in the species diversity of these genera.
Key words: fungal evolution, Helianthemum, internal transcribed spacer, mycorrhizal fungi
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). 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
).
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). 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 Tuberales 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Variability in spore ornamentation was the most important morphological feature for discriminating between these species. A comparative table of major morphological features is provided (Table II
).
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). 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-trimethyl-ammonium 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 µL 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 1x Tris-borate-EDTA buffer. The gels were stained with ethidium bromide and photographed under ultraviolet light. 
x 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
).
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). 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 DAB = -(3/4)ln[1 - 4/3(Su/I + Su)][1 - G/T] + G/T. DAB 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
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. 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 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 ).
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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 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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). 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 tramal 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 re-establishment.
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 Terfezia 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 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 well-supported 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. leptoderma. 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 determine the genetic structure of Terfezia and Tirmania populations.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication August 7, 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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