The diversity of Terfezia desert truffles: new species and a highly variable species complex with intrasporocarpic nrDNA ITS heterogeneity

Desert truffles belonging to Terfezia are well known mycorrhizal members of the mycota of the Mediterranean region and the Middle East. We aimed to test (i) whether the morphological criteria of Terfezia species regularly collected in Spain enable their separation and (ii) whether the previously hypothesized edaphic/biotic specificity of one group could be confirmed by study of a larger number of specimens. The species T. arenaria and T. claveryi can be identified unambiguously by morphological characters. We consider T. leptoderma as a distinct species while several lineages of similar spiny spored Terfezia truffles with cellular peridium were detected that have no obvious anatomical differences. Several species treated generally as synonyms of T. olbiensis have been described in this group, and because they cannot be unambiguously assigned to separate lineages we propose to consider the group as the T. olbiensis species complex. A high level of intrasporocarpic variation of the nrDNA ITS was detected in the T. olbiensis species complex, especially in one of its lineages. We detected no exclusive specificity to either plant associates or soil, except in T. leptoderma, which was associated with Quercus spp. and cistaceous plants on acidic soils. Nevertheless the clades showed a tendency either to associate with Quercus/Helianthemum/Cistus or Pinus hosts. Specimens having distinct anatomical features, reticulate spores and cellular peridium formed a separate group in the molecular phylogenetic analyses of nrDNA ITS and LSU regions; for these specimens we propose a new species, Terfezia alsheikhii sp. nov.

[1]  T. Ward,et al.  Evolution of a large ribosomal RNA multigene family in filamentous fungi: birth and death of a concerted evolution paradigm. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[2]  M. P. Cummings,et al.  PAUP* Phylogenetic analysis using parsimony (*and other methods) Version 4 , 2000 .

[3]  J. Heilmann‐Clausen,et al.  Population Evidence of Cryptic Species and Geographical Structure in the Cosmopolitan Ectomycorrhizal Fungus, Tricholoma scalpturatum , 2008, Microbial Ecology.

[4]  A. Morte,et al.  Biotechnology and Cultivation of Desert Truffles , 2008 .

[5]  T. Horton.  Molecular approaches to ectomycorrhizal diversity studies: variation in ITS at a local scale , 2002, Plant and Soil.

[6]  K. O’Donnell,et al.  Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. , 1997, Molecular phylogenetics and evolution.

[7]  A. Varma.  Mycorrhiza : state of the art, genetics and molecular biology, eco-function, biotechnology, eco-physiology, structure and systematics , 2008 .

[8]  J. Trappe.  A synopsis of the carbomycetaceae and terfeziaceae (tuberales) , 1971 .

[9]  J. Trappe,et al.  Phylogenetic studies of Terfezia pfeilii and Choiromyces echinulatus (Pezizales) support new genera for southern African truffles: Kalaharituber and Eremiomyces. , 2005, Mycological research.

[10]  F. Martin,et al.  Polymorphism at the ribosomal DNA ITS and its relation to postglacial re-colonization routes of the Perigord truffle Tuber melanosporum. , 2004, The New phytologist.

[11]  J. Trappe,et al.  Desert Truffles of the Australian Outback: Ecology, Ethnomycology, and Taxonomy , 2008, Economic Botany.

[12]  G. Kovács,et al.  In Vitro interaction of the truffleTerfezia terfezioides withRobinia pseudoacacia andHelianthemum ovatum , 2008, Folia Microbiologica.

[13]  J. Huelsenbeck,et al.  MRBAYES : Bayesian inference of phylogeny , 2001 .

[14]  J. Rosselló,et al.  Better the devil you know? Guidelines for insightful utilization of nrDNA ITS in species-level evolutionary studies in plants. , 2007, Molecular phylogenetics and evolution.

[15]  J. Thompson,et al.  The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. , 1997, Nucleic acids research.

[16]  M. Nei,et al.  MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. , 2007, Molecular biology and evolution.

[17]  D. Filatov.  proseq: A software for preparation and evolutionary analysis of DNA sequence data sets , 2002 .

[18]  F. D. Calonge,et al.  Nuevos datos sobre los hongos hipogeos de España. II. Géneros Balsamia, Delastria y Genea, novedades para el catálogo español , 1985 .

[19]  Kathryn F. Beal,et al.  The Staden package, 1998. , 2000, Methods in molecular biology.

[20]  Amer. Montecchi,et al.  Funghi ipogei d'Europa , 2000 .

[21]  Andrew W. Claridge,et al.  Comparative taxonomy of desert truffles of the Australian outback and the African Kalahari , 2010, Mycological Progress.

[22]  John P. Huelsenbeck,et al.  MrBayes 3: Bayesian phylogenetic inference under mixed models , 2003, Bioinform..

[23]  V. Kagan-Zur,et al.  Two ITS forms co-inhabiting a single genet of an isolate of Terfezia boudieri (Ascomycotina), a desert truffle , 2004, Antonie van Leeuwenhoek.

[24]  A. Morte,et al.  Partial purification, characterisation and histochemical localisation of alkaline phosphatase from ascocarps of the edible desert truffle Terfezia claveryi Chatin. , 2009, Plant biology.

[25]  O. Gascuel,et al.  A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. , 2003, Systematic biology.

[26]  G. Mueller,et al.  A ‘dirty’ business: testing the limitations of terminal restriction fragment length polymorphism (TRFLP) analysis of soil fungi , 2006, Molecular ecology.

[27]  Austen R. D. Ganley,et al.  Highly efficient concerted evolution in the ribosomal DNA repeats: total rDNA repeat variation revealed by whole-genome shotgun sequence data. , 2007, Genome research.

[28]  John P. Huelsenbeck,et al.  MRBAYES: Bayesian inference of phylogenetic trees , 2001, Bioinform..

[29]  Kazutaka Katoh,et al.  Improved accuracy of multiple ncRNA alignment by incorporating structural information into a MAFFT-based framework , 2008, BMC Bioinformatics.

[30]  K. Hansen,et al.  Truffle trouble: what happened to the Tuberales? , 2007, Mycological research.

[31]  D. Posada.  jModelTest: phylogenetic model averaging. , 2008, Molecular biology and evolution.

[32]  Miguel Verdú,et al.  Ecological interactions are evolutionarily conserved across the entire tree of life , 2010, Nature.

[33]  D. Rizzo,et al.  Intra-specific and intra-sporocarp ITS variation of ectomycorrhizal fungi as assessed by rDNA sequencing of sporocarps and pooled ectomycorrhizal roots from a Quercus woodland , 2007, Mycorrhiza.

[34]  Yong-Fang Li,et al.  Cryptic species in the Terfezia boudieri complex , 2009, Antonie van Leeuwenhoek.

[35]  F. Martin,et al.  Molecular phylogeny of the mycorrhizal desert truffles (Terfezia and Tirmania), host specificity and edaphic tolerance , 2002, Mycologia.

[36]  K. Ko,et al.  Three nonorthologous ITS1 types are present in a polypore fungus Trichaptum abietinum. , 2002, Molecular phylogenetics and evolution.

[37]  Michael P. Cummings,et al.  PAUP* [Phylogenetic Analysis Using Parsimony (and Other Methods)] , 2004 .

[38]  J. Trappe,et al.  Desert Truffles of the African Kalahari: Ecology, Ethnomycology, and Taxonomy , 2008, Economic Botany.

[39]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[40]  Y. Sitrit,et al.  Differential expression of fungal genes at preinfection and mycorrhiza establishment between Terfezia boudieri isolates and Cistus incanus hairy root clones. , 2006, The New phytologist.

[41]  H. Kauserud,et al.  Ribosomal DNA variation, recombination and inheritance in the basidiomycete Trichaptum abietinum: implications for reticulate evolution , 2003, Heredity.

[42]  J. Trappe,et al.  Imaia, a new truffle genus to accommodate Terfezia gigantea , 2008, Mycologia.

[43]  M. Weiß,et al.  Intragenomic variation of fungal ribosomal genes is higher than previously thought. , 2008, Molecular biology and evolution.

[44]  Z. Pénzes,et al.  Molecular study of arbuscular mycorrhizal fungi colonizing the sporophyte of the eusporangiate rattlesnake fern (Botrychium virginianum, Ophioglossaceae) , 2007, Mycorrhiza.

[45]  K. Katoh,et al.  MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. , 2002, Nucleic acids research.

[46]  K. Winka,et al.  Alternative Methods of extracting and Amplifying Dna from lichens , 2000, The Lichenologist.

[47]  M. Janex-Favre,et al.  L'ascocarpe hypogé d'une Terfez française (Terfezia leptoderma Tul., Tuberales, Discomycètes) , 1988 .