The Diversity of Terfezia Desert Truffles: New Species and a Highly Variable Species Complex with Intrasporocarpic Nrdna ITS Heterogeneity
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Mycologia, 103(4), 2011, pp. 841–853. DOI: 10.3852/10-312 # 2011 by The Mycological Society of America, Lawrence, KS 66044-8897 The diversity of Terfezia desert truffles: new species and a highly variable species complex with intrasporocarpic nrDNA ITS heterogeneity Ga´bor M. Kova´cs1 INTRODUCTION Tı´mea K. Bala´zs Desert truffles are hypogeous ascomycetes living on Department of Plant Anatomy, Institute of Biology, Eo¨tvo¨s Lora´nd University, Pa´zma´ny Pe´ter se´ta´ny 1/C, several continents (Dı´ez et al. 2002, Ferdman et al. H-1117 Budapest, Hungary 2005, Trappe et al. 2010a) where they play important roles as mycorrhizal partners of plants and their fruit Francisco D. Calonge bodies are a potentially important food source Marı´a P. Martı´n (Trappe et al. 2008a, b). A study revealed that fungi Departamento de Micologı´a, Real Jardı´n Bota´nico, adapted to deserts evolved in several lineages of the CSIC, Plaza de Murillo 2, 28014 Madrid, Spain Pezizaceae (Trappe et al. 2010a). Among the genera in these lineages Terfezia represents the best known and probably most frequently collected desert truffles Abstract: Desert truffles belonging to Terfezia are (Dı´ez et al. 2002, Læssøe and Hansen 2007). well known mycorrhizal members of the mycota of the Although several members of the genus were de- Mediterranean region and the Middle East. We aimed scribed from different continents, recent molecular- to test (i) whether the morphological criteria of taxonomic studies revealed that probably only the Terfezia species regularly collected in Spain enable species from the Mediterranean region and the their separation and (ii) whether the previously Middle East belong in Terfezia s. str. The genera hypothesized edaphic/biotic specificity of one group Imaia (Kova´cs et al. 2008) and Kalaharituber (Ferd- could be confirmed by study of a larger number of man et al. 2005) were introduced to accommodate specimens. The species T. arenaria and T. claveryi can respectively Terfezia gigantea Imai from North Amer- be identified unambiguously by morphological char- ica and Japan and Terfezia pfeilii Henn. from South acters. We consider T. leptoderma as a distinct species Africa. The American Terfezia longii Gilkey and T. while several lineages of similar spiny spored Terfezia spinosa Harkn. also belong in different genera truffles with cellular peridium were detected that have (Kova´cs et al. 2011), while Terfezia austroafricana no obvious anatomical differences. Several species Marasas & Trappe from South Africa belongs in treated generally as synonyms of T. olbiensis have Mattirolomyces (Trappe et al. 2010a, b). been described in this group, and because they Five Terfezia species have been reported regularly cannot be unambiguously assigned to separate from the Mediterranean region and the Middle East; lineages we propose to consider the group as the T. they are T. arenaria (Moris) Trappe, T. boudieri olbiensis species complex. A high level of intraspor- Chatin, T. claveryi Chatin, T. leptoderma Tul. and T. ocarpic variation of the nrDNA ITS was detected in olbiensis Tul. & C. Tul. (Montecchi and Sarasini 2000). the T. olbiensis species complex, especially in one of Additional Terfezia species have been described from its lineages. We detected no exclusive specificity to the region but are treated mostly as synonyms of these either plant associates or soil, except in T. leptoderma, five species. Four of the five are common in Spain; the which was associated with Quercus spp. and cistaceous fifth, T. boudieri, commonly collected in North Africa, plants on acidic soils. Nevertheless the clades showed Middle East and southwestern Asia, also has been a tendency either to associate with Quercus/Helianthe- reported from the Iberian Peninsula (Moreno et al. mum/Cistus or Pinus hosts. Specimens having distinct 2002). The true Terfezia species are mycorrhizal and anatomical features, reticulate spores and cellular have been used for mycorrhizal experiments with peridium formed a separate group in the molecular plants in the Cistaceae, mainly Helianthemum spp. (see phylogenetic analyses of nrDNA ITS and LSU regions; references in Kova´cs et al. 2003). Plantations of for these specimens we propose a new species, inoculated hosts have been established for cultivation Terfezia alsheikhii sp. nov. of Terfezia species with economic importance (Morte Key words: ascomycota, ascospores, concerted et al. 2008). Terfezia claveryi Chatin and T. boudieri evolution, host specificity, ITS variability, Pezizaceae have been used as model organisms for molecular biological studies (Morte et al. 2008, Navarro-Ro´denas et al. 2009, Zaretsky et al. 2006). Submitted 5 Oct 2010; accepted for publication 17 Jan 2011. Terfezia arenaria, T. claveryi and T. boudieri can be 1 Corresponding author. E-mail: [email protected] easily separated by morphological characters, whereas 841 842 MYCOLOGIA the separation of T. olbiensis and T. leptoderma are less When intrasporocarpic ITS variability was detected clear, judging from keys to species of the genus during the verification of electropherograms after direct (Montecchi and Sarasini 2000, Alsheikh 1994). sequencing (see below) the ITS region was amplified again, Terfezia olbiensis and T. leptoderma and the other but with a high fidelity Pfu DNA polymerase (MBI spiny spored Terfezia species have been considered as Fermentas, Vilnius, Lithuania). Before cloning into a pGEMT Easy Vector system (Promega, Madison, Wisconsin) different developmental stages and treated as syno- the amplicons purified after gel electrophoresis (Gel-M Kit, nyms by several authors (see DISCUSSION). Viogene, Hong-Kong) were A-tailed with a normal Taq Dı´ez et al. (2002) studied Mediterranean Terfezia polymerase and dATP (MBI Fermentas, Vilnius, Lithuania) species by both morphological and molecular and purified again. The plasmids were transformed into phylogenetic methods. The ITS regions of 11 chemically competent JM109 E. coli cells (Promega, specimens of four species were sequenced and Madison, Wisconsin) following manufacturer’s instructions. analyzed (Dı´ez et al. 2002). They found considerable Positive clones were selected and purified for sequencing as variation of the ITS sequences of those T. leptoderma described by Kova´cs et al. (2007). Twelve positive clones of specimens and hypothesized this variation might each amplicon were sequenced by LGC Genomics (Berlin, relate to edaphic and/or biotic factors such as soil Germany) with universal primers. We deposited the LSU types and/or host plants. Nevertheless because of and ITS sequences obtained either from the clones or the the small number of specimens studied the question directly sequenced amplicons in GenBank (HQ698054– HQ698149, SUPPLEMENTARY TABLE I). was left unresolved. Intrahyphal, heterokaryotic variation of the nrDNA was found in T. boudieri Phylogenetic analyses.—Sequences were compared with (Aviram et al. 2004) and intraspecific genetic sequences deposited in public databases by BLASTn variability was interpreted as a cryptic speciation in (Altshul et al. 1990; http://www.ncbi.nih.gov/blast/). LSU the taxon (Ferdman et al. 2009). sequences were included in a Pezizaceae dataset (Læssøe We studied Terfezia specimens deposited in the and Hansen 2007, Trappe et al. 2010a) to analyze the Mycological Collection of the Herbarium of the Real position of the new species. Several datasets of ITS Jardı´n Bota´nico, Madrid, (MA-Fungi) to test whether sequences were analyzed. Here we present the results of two of them. Group 1 contains all ITS sequences except the (i) the morphological criteria of species regularly cloned sequences of ITS types from one ascoma, plus ITS collected in Spain enable their unambiguous separa- sequences of Terfezia species deposited in GenBank, and tion and (ii) the previously hypothesized background Peziza depressa as outgroup. Group 2 contains all ITS of the intraspecific variation of the ITS region of sequences and ITS sequence types of the T. leptoderma-T. nrDNA could be confirmed by study of a larger olbiensis group with T. arenaria and T. claveryi ITS number of specimens. In addition, some specimens sequences as outgroup. In this dataset we used only the with distinctive anatomical features found during this sequences obtained in the present study. research formed a distinct group in the molecular Sequences were aligned with Clustal X (Thompson et al. phylogenetic analyses; for these we propose a new 1997) and MAFFT (Katoh et al. 2002, Katoh and Toh 2008). Terfezia species. Alignments were checked and adjusted manually with ProSeq 2.9 (Filatov 2002) and deposited in TreeBASE (S11098). The best-fit nucleotide substitution model was MATERIALS AND METHODS selected with the program jModelTest (Posada 2008) applying the Akaike information criterion (AIC). This Microscopic study.—Examination of dried specimens by model was used to calculate distances for a neighbor light microscopy was conducted with rehydrated freehand joining (NJ) analyses with the PAUP* 4.0b10 software sections cut from ascomata. Cell, spore and ascus sizes were package (Swofford 2003). We verified branches with a NJ measured with the aid of an ocular micrometer. Micro- bootstrap (NJB) analysis with 1000 replicates. Phylogenies graphs were obtained with Nikon microscopes equipped also were inferred by parsimony analyses with PAUP with digital cameras. Ascospores were studied by scanning heuristic search for the most parsimonious (MP) trees. electron microscopy (Hitachi S-3000N SEM) after gold