Antonie van Leeuwenhoek (2010) 98:429–436 DOI 10.1007/s10482-010-9456-y ORIGINAL PAPER Phylogenetic affiliation of the desert truffles Picoa juniperi and Picoa lefebvrei Imed Sbissi • Mohamed Neffati • Abdellatif Boudabous • Claude Murat Maher Gtari • Received: 26 January 2010 / Accepted: 12 May 2010 / Published online: 18 June 2010 Ó Springer Science+Business Media B.V. 2010 Abstract The molecular phylogeny and comparative morphological studies reported here provide evidence for the recognition of the genus Picoa, an hypogeous desert truffle, in the family Pyronemataceae (Ascomycota, Pezizales). Picoa juniperi and Picoa lefebvrei were reassigned to the genus Picoa based on large subunit (LSU) sequence (28S) rDNA and internal transcribed spacer (ITS) rDNA (including the partial 18S, ITS1, ITS2, 5.8S gene, and partial 28S of the nuclear rDNA) data. Morphological studies of spores, asci, perida, and gleba revealed high similarities between P. lefebvrei and P. juniperi, thereby confirming the membership of both species in the genus Picoa. These two species were primarily distinguishable based on ascospore ornamentation. Keywords Pyronemataceae
Picoa juniperi
Picoa lefebvrei
Molecular phylogeny
ITS
LSU rDNA
Morphology I. Sbissi
A. Boudabous
M. Gtari (&) Laboratoire Microorganismes and Biomolécules Actives, Département de Biologie, Faculté des Sciences de Tunis, Campus Universitaire, 2092 Tunis, Tunisia e-mail: maher.gtari@fst.rnu.tn I. Sbissi
M. Neffati Laboratoire d’Ecologie Pastorale, Institut des Régions Arides, 4119 Médenine, Tunisia C. Murat UMR INRA-UHP ‘Interactions ArbresMicroorganismes’, INRA-Nancy, Champenoux, France Introduction Picoa is hypogeous desert truffle (Ascomycetes) that has been documented in areas extending the Mediterranean to Middle East arid lands (Al-Scheikh and Trappe 1983; Moreno et al. 2000a, b; Ammarellou and Trappe 2007). It is presumed to establish a mutualistic association with roots of annual and perennial herbaceous plants of the Helianthemum genus (Gutiérrez et al. 2003; Slama et al. 2006). Five Picoa species are recorded in the Index Fungorum: P. carthusiana, P. juniperi, P. lefebvrei, P. melospora, and P. pachyascus. Unlike other truffles, the taxonomic status of the genus Picoa and the membership of the five proposed species in this genus remain uncertain, and several widely divergent taxonomic outlines have been reported. Vittadini (1831) was the first to propose the generic name Picoa and assign this truffle to the Tuberaceae family based on the type species P. juniperi. It was subsequently transferred from Tuberaceae to Terfeziaceae by Fischer (1897) and then to Balsamiaceae by Trappe (1979). P. lefebvrei was originally described as Phaeangium lefebvrei in 1894 by Patouillard who considered P. lefebvrei as the holotype species for the genus Phaeangium, but several other authors later considered this species to be member of the genus Picoa (Maire 1906; Moreno et al. 2000a, b). P. carthusiana, originally described by Tulasne and Tulasne (1862), was reassigned to Leucangium carthusianum by Trappe (1971) based on ascocarp differences with P. juniperi. Morphological 123 430 Antonie van Leeuwenhoek (2010) 98:429–436 and molecular data have provided evidence for the membership of L. carthusianum within the Morchellaceae–Helvellaceae lineage (Li 1997; O’Donnell et al. 1997), while its exact assignment still remains uncertain (Læssøe and Hansen 2007). P. pachyascus, originally described by Lange (1956), was recently documented as a synonym of Imaia gigantean in Morchellaceae (Kovacs et al. 2008). P. melospora was described by Moreno et al. (2000a, b) from the Iberian Peninsula. This species, which has not yet been phylogenetically characterized, presents unusual morphological features for the genus, namely, one to five spores per ascus and elongated and smooth ascospores. Preliminary large subunit (LSU) rDNA sequence data suggest a close relationship between P. juniperi and Otidea spp. within the Pyrenomataceae family (O’Donnell et al. 1997). More recently, Tedersoo et al. (2010) considered Picoa to be a member of the Geopora lineage, but the exact phylogenetic positions of P. juniperi and P. lefebvrei have not yet been assessed. In the study reported here, ascocarps morphologically characterized as those from P. juniperi and P. lefebvrei, respectively, were collected from the Tunisian arid lands. Based on sequence data on two genomic regions, we have confirmed that Picoa belongs to Pyronemataceae. Phylogenetic analyses revealed that this genus is closely related to Geopora. Materials and methods Origin of the samples Fruit bodies were collected from the Medenine region in southern Tunisia (Table 1). This area is situated in a low-aridity bio-climatic zone with an average annual precipitation of 180 mm, a low average annual temperature of 19.9°C, and a mild winter. The average minimum temperature of the coldest month is 7°C, and the mean maximum temperature of the warmest months is 50°C. The ascocarps were freshly harvested, superficially disinfected by shaking in 30% (v/v) H2O2 for 5 min. and aseptically rinsed several times with sterile water to eliminate any possibly trapped pocket of soil and microorganisms. The gleba was cut into small pieces kept at -80°C in sterile petri dishes before being freeze-dried overnight in a lyophilizator (FLEXI-Dry; FTS Systems, Milton, MA). Some samples were freshly used for microscopic observations. All samples have been deposited in the mycological specimen collection of the Royal Botanic Gardens Kew under the accession K(M)165772. Morphological analysis All fruit bodies were macro- and micro-morphologically characterized. Sections were mounted in 5% Table 1 Collection of fruit bodies sequenced in this study Species Geographic origin Host plant IRA-MBA Herbarium accessiona GenBank LSU accession number GenBank ITS accession number Picoa juniperi Medenine Helianthemum sessiliflorum IRA-MBAsb1 GU391549 GU391559 P. juniperi Medenine H. sessiliflorum IRA-MBAsb2 GU391550 GU391560 P. juniperi Medenine H. sessiliflorum IRA-MBAsb3 GU391551 GU391561 P. juniperi Medenine H. sessiliflorum IRA-MBAsb4 GU391552 GU391562 P. lefebvrei Medenine H. sessiliflorum IRA-MBAsb5 GU391553 GU391563 P. juniperi Medenine H. sessiliflorum IRA-MBAsb6 GU391558 GU391564 P. juniperi Medenine H. sessiliflorum IRA-MBAsb7 GU391554 GU391565 P. juniperi Medenine H. sessiliflorum IRA-MBAsb8 GU391555 GU391566 P. juniperi Medenine H. sessiliflorum IRA-MBAsb9 GU391556 GU391567 P. juniperi Medenine H. sessiliflorum IRA-MBAsb10 ND GU391568 P. lefebvrei Medenine H. sessiliflorum IRA-MBAsb11 GU391548 GU391570 P. juniperi Medenine H. sessiliflorum IRA-MBAsb12 GU391557 GU391569 a K(M)165772 is the accession in the mycological specimen collection of the Royal Botanic Gardens Kew ITS, Internal transcribed spacer; LSU, large subunit; ND, not determined 123 Antonie van Leeuwenhoek (2010) 98:429–436 KOH and cotton blue–lactophenol and observed under a light microscope at 10009 magnitude. KOH was not used for spore measurement because alkaline solutions may dissolve the spore ornaments (Ferdman et al. 2005). Cubes and slices, including the peridium and gleba, from fresh fruit bodies were embedded in paraffin, sectioned, and mounted for light microscopy. For scanning electron microscopy (SEM), dried material of the ascoma was dehydrated on a glass slide, then post fixed in osmium tetroxide, washed in phosphate buffer (pH 7.2), dehydrated first stepwise in ethanol (20–99%) and then in pure acetone, air-dried coated with gold–palladium, and examined using in a scanning electron microscope (Quanta 200; FEI, Hillsboro, OR). DNA extraction, PCR amplification, and sequencing DNA extraction was carried out on approximately 50 mg of freeze-dried fruit bodies. Tissues of the gleba were ground in liquid nitrogen. and nucleic acids were extracted according to the method of Henrion et al. (1994). DNA was resuspended in 50 ll of TE buffer (10 mM Tris-HCl pH 7.4; 1 mM of EDTA) and stored at -20°C. The internal transcribed spacer (ITS) and the 50 LSU regions of the nuclear rDNA were separately amplified using the following primer pairs: ITS1 (50 -TCCGTAGGTGAACCTGCGG-30 ) and ITS4 (50 -TCCTCCGCTTATTGATATGC- 30 ) for the ITS rDNA; LROR (50 -ACCCGCTGAACTTAAGC-30 ) and LR7 (50 -TACTACCACCAAGATCT-30 ) for the 50 LSU rDNA (Vilgalys and Hester 1990; White et al. 1990). The amplification reactions were performed in a 50-ll volume of reaction mixture [1 mM of each primer, 0.2 mM of each dNTP, and 2.5 U of Taq polymerase (Promega, Madison, WI] in a DNA thermal cycler (2400 geneAmp PCR thermocycler; Perkin Elmer, Foster City, CA). The cycling conditions were: an initial denaturation at 95°C for 2 min, followed by 35 cycles of a 1-min denaturation at 94°C, a 40-s annealing at either 53°C (ITS rDNA) or 47°C (LSU, 28S rDNA), and a 1-min elongation at 72°C, with a final elongation step at 72°C for 10 min. Amplification products were analyzed in 1.5% agarose gel in 0.59 TBE buffer (89 mmol l-1 Tris, 89 mmol l-1 borate, 2 mmol l-1 EDTA), stained with ethidium bromide, and visualized under UV light (Sambrook et al. 1989). The PCR products were 431 purified with QIAquick Wizard PCR purification Kit (Promega) according to the manufacturer’s instructions, and the sequences were determined by cycle sequencing using the Taq Dye Deoxy Terminator Cycle Sequencing kit (Applied Biosystems; HTDS, Tunisia) and fragment separation in an ABI PrismTM 3130 DNA sequencer (Applied Biosystems; HTDS, Tunisia). Sequence analysis The 28S LSU and ITS rDNA nucleotide sequences were aligned using ClustalW (Thompson et al. 1997), and the alignment was manually edited with MEGA 4.0 (Tamura et al. 2007). Using RAxML (Stamatakis et al. 2005), we constructed a maximum-likelihood cladogram with 1000 fast bootstraps by following the GTR ? G base substitution model using Neolecta vittelina and Tarzetta catinus sequences as the outgroup for the LSU and ITS analyses, respectively. The tree was edited with FigTree (Rambaut 2008). In parallel, a Bayesian inference was realized with MrBayes (Ronquist and Huelsenbeck 2003) using the GTR ? G model and 1,000,000 generations. The sequences for P. juniperi and P. lefebvrei 28S LSU and ITS rDNA were submitted to GenBank under the accession numbers listed in Table 1. Results Morphological analysis The Picoa fruiting bodies collected in this study (locally called ‘‘Zouber’’) appear in early February, when rainfall is adequate, at a soil depth close to 5 cm and near host plants (H. sessiliflorum). Fruiting bodies of P. juniperi were morphologically characterized (Fig. 1). The ascomata are 1–3 cm in size and very light in appearance; they have irregular forms and are often associated in clusters of four individuals. The peridium (Fig. 1a) has irregular pyramidal warts (and was more distinctly warty when dried) and is light brown to dark brown when young and blackish brown at maturity (Fig. 1b). The gleba is generally white, with fertile tissue separated by sterile veins (Fig. 1c). Asci (Fig. 1d and e) are of various shapes, double layered, hyaline, and thin walled and contain six to eight oval ascospores that are smooth 123 432 Fig. 1 Light and scanning electron micrographs of Picoa juniperi ascomata. a Warty peridium, bar 1 mm, b cross section of the gleba and peridium, bar 1 mm, c cross section of the gleba with sterile veins (arrowheads), bar 0.5 mm), d asci in cotton blue– lactophenol, bar 10 lm, e asci in 5% KOH, bar 10 lm, f mature ascospores showing a typical large lipid bodies (l), bar 10 lm, g smooth spore (in scanning electron microscopy), bar 10 lm. The white gleba appear darker in c due to the quality of the preparation (Color figure online) 123 Antonie van Leeuwenhoek (2010) 98:429–436 Antonie van Leeuwenhoek (2010) 98:429–436 433 Table 2 Characterization of Picoa, Leucangium, and Geopora genera Characteristics Picoa juniperi Picoa lefebvreia Leucangium carthusianumb Geopora spc Ascoma Hypogeous with irregular forms, stereothecia Hypogeous, gregarious, sub-globose, stereothecia Hypogeous, stereothecia Epigeous, several species have a small subterranean apothecia, ptychotheciae Peridium Warty, brown to dark brown Reddish brown to dark brown with irregular pyramidal rounded warts. Dark with medullary excipulum with cylindrical to isodiametric cells Gleba White, crumbly, with fertile Off-white, very crumbly, with fertile pockets pockets separated by sterile separated by sterile vein clearly distinguished veins. Dark, very crumbly with None fertile pockets separated by sterile veins Spore shape Oval Outer surface irregular to warted or furrowed, covered with dark hairs Oval Lemon shaped Subglobose, ellipsoid Spore Smooth ornamentation Warty Smooth Smooth Host plant Cistaceae Helianthemum sp.d Pseudotsuga menziesii Wide range of hosts Pinaceaed a Cistaceae Helianthemum sessiliflorumd According to Patouillard (1894), Maire(1906), Moreno et al. (2000a, b), and Gutiérrez et al. (2003) b According to Patouillard (1894), Maire (1906), Li (1997), and Palfner and Agerer (1998) c According to Harold and Burdsall (1965, 1968); Jack and Gaud (1997). and Wei et al. (2010) d Preferred host plant e Except for Geopora cooperi and G. clausa. Læssøe and Hansen 2007; Smith and Healy 2009 and contain a dark lipid body at maturity (Fig. 1f, g). Morphological features of P. juniperi, P. lefebvrei, and L. carthusianum are listed in Table 2. Phylogenetic analysis Analysis of the 50 end of the LSU rDNA region (including the D1 and D2 domains) data set of 36 Pezizalean fungi, including the 11 specimens (from IRA-MBAsb1 to IRA-MBAsb11) sequenced in this, was performed using maximum-likelihood and Bayesian methods. The topologies of the phylogenetic trees built with maximum likelihood and Bayesian inference were similar and clearly indicate that Picoa is a member of the Pyronemataeae. The 11 Picoa specimens, which share 95–99% sequence identity and form a coherent cluster well supported by significant bootstrap and posterior probability values (86–100%), are most closely related to Geopora species (IRA-MBAsb11 shows the highest sequence identity; 97% with Geopora cooperi DQ220342). Notwithstanding, Geopora spp. differ from Picoa spp. on the basis of the development mode, forms, spore discharge, and the associative host plant (Table 2). Noticeably, Leucangium carthusianum (synonym Picoa carthusiana) was reliably placed in the Morchellaceae lineage (78% similarity in base pairs) and shares 95% LSU sequence identity with Imaia gigantea (synonym of Picoa pachyascus) (Fig. 2). These results were further tested and confirmed based on the phylogeny inferred from the analysis of the ITS rDNA sequences. Moreover, the generated phylogenetic tree (Fig. 3) supports the separation of the genus Picoa into two clusters with significant bootstrap and posterior probability values (92–100%): (1) cluster one associating P. juniperi specimens and (2) cluster two regrouping P. lefebvrei specimens. Discussion The microscopic comparisons performed in this study and in previous studies reveal that there are distinct morphological links between P. juniperi and Phaeangium lefebvrei that can allow them to be recognized as members of the same genus. The main difference between these two species is the ascospore ornamentation. Using specimens collected in Kuwait, 123 434 Antonie van Leeuwenhoek (2010) 98:429–436 91 100 89 74 98 100 87 100 100 100 86 100 62 85 100 100 100 100 100 99 99 100 85 100 50 89 100 100 100 100 75 100 100 91 99 61 97 71 100 100 Morchellaceae-Discinaceae 100 100 Geopora spp. 93 100 100 100 Pyronemataceae 98 100 77 IRA-MBAsb7 IRA-MBAsb6 IRA-MBAsb1 IRA-MBAsb8 Picoa juniperi IRA-MBAsb9 IRA-MBAsb11 IRA-MBAsb2 IRA-MBAsb4 IRA-MBAsb3 IRA-MBAsb5 Picoa lefebvrei IRA-MBAsb12 Geopora cooperi (DQ220341) Geopora cooperi (DQ220340) Geopora clausa (DQ220339) Geopora cf. (DQ220344) Geopora arenicola (DQ220337) Geopora arenicola (DQ220336) Geopora sp. (DQ220345) Tricharina ochroleuca (DQ220445) Tricharina praecox (DQ646525) Tricharina gilva (DQ220444) Miladina lecithina (DQ220372) Miladina lecithina (DQ220371) Ramsbottomia asperior (DQ220407) Ramsbottomia asperior (DQ220408) Ramsbottomia asperior (DQ220406) Otidea sp. FJ404767 Otidea sp. (FJ404766) Leucangium carthusianum (GQ379720) Leucangium carthusianum (U42674) Disciotis venosa (AJ698472) Verpa conica (AY544666) Morchella esculenta (AY533016) Imaia gigantea (EU327201) Imaia gigantea (EU327203) Imaia gigantea (EU327202) Neolecta vitellina (DQ470985) Fig. 2 Maximum-likelihood cladogram inferred from the 787bp large subunit (LSU) region alignment, demonstrating the placement of Picoa within Pyrenomataceae. Bootstrap values [50 are shown above branches, and posterior probability values [50 are shown below branches. The phylogenetic trees were built with maximum likelihood and Bayesian inferences are topologically similar Iraq, North Africa (Al-Scheikh and Trappe 1983), and Tunisia (data not shown), we have shown that P. juniperi spores are oval and smooth while those of P. lefebrei appear warty and ornamented at maturity. The molecular positioning of L. carthusianum (syn. Picoa carthusiana) within the Morchellaceae–Discinaceae lineage, proposed in this study, corroborates earlier morphological and ecological data and confirms the exclusion of this species from the genus Picoa (O’Donnell et al. 1997). An ultra-structural study of L. carthusianum performed by Li (1997) showed that this species is characterized by lemon- shaped and multinucleated ascospores. This species has been reported in Europe and North America (Trappe 1971) in mutualistic association with the forest tree Pseudotsuga menziesii (Palfner and Agerer 1998). Ribosomal DNA analyses have enabled the genus Picoa to be assigned to the Pyronemataceae and to confirm that Picoa is closely related to Geopora (Tedersoo et al. 2010). The phylogenetic position of P. juniperi and P. lefebrei proposed in this report is well supported by morphological and ecological features. The two Picoa species are hypogeous taxa 123 Antonie van Leeuwenhoek (2010) 98:429–436 435 IRA-MBAsb3 IRA-MBAsb4 67 IRA-MBAsb7 IRA-MBAsb1 IRA-MBAsb10 IRA-MBAsb8 66 Picoa juniperi IRA-MBAsb11 IRA-MBAsb6 98 100 IRA-MBAsb2 52 97 100 IRA-MBAsb9 IRA-MBAsb5 99 100 95 100 IRA-MBAsb12 P. lefebvrei (GQ228095) P. lefebvrei (GQ228092) 99 100 87 100 Picoa lefebvrei P. lefebvrei (AF387654) 100 97 P. lefebvrei (AF387653) P. lefebvrei (AF387652) Geopora cooperi (GU184101) 88 90 Uncultured Tricharina (EU726331) 65 78 80 95 Uncultured Tricharina (EU726332) Geopora cf. cooperi (FJ789596) Geopora cervina (FM206458) 88 99 Geopora spp. Geopora tenuis (FM206429) 56 71 Geopora sepulta (FM206476) 98 100 Geopora foliacea (FM206469) 100 100 100 100 Geopora arenicola (FM206472) Otidea subterranea (FJ404767) Otidea subterranea (FJ404766) Aleuria aurantia (AF072090) 81 75 89 95 Scutellinia scutellata (FJ235141) Scutellinia colensoi (AY220830) Tarzetta catinus (FM206478) Fig. 3 Maximum-likelihood cladogram based on the 413-bp internal transcribed spacer (ITS) region alignment of Picoa, Geopora, and related Pyrenomataceae species. Bootstrap values [50 are shown above branches, and posterior probability values [50 are shown below branches. Topologies of the phylogenetic tree with maximum likelihood and Bayesian inferences are similar with white to off-white and crumbly gleba (stereothecia) and the absence of forcible spore discharge. Geopora species possess hollow ascocarps (ptychothecia), are epigeous or partially hypogeous (hypogeous in their early stages of development, except for Geopora cooperi and G. clausa, which are mostly hypogeous; Læssøe and Hansen 2007; Smith and Healy 2009), and emerge at the ground surface at maturity with few (if any) convolutions and a functional operculum that opens at the ground surface (Harold and Burdsall 1965, 1968). The ascospores of these Geopora species are discharged through an operculum. Picoa species are also divergent from Geopora spp. based on the host plants and the geographic distribution. P. juniperi and P. lefebrei occur in the Mediterranean region and in Middle East arid lands, and they are associated with members of Cistaceae and, preferentially, with Helianthemum species (Moreno et al. 2000a, b; Gutiérrez et al. 2003; Slama et al. 2006). In contrast, Geopora spp. are associated with a wide range of host plants that are essentially found in Pinaceae forest stands (Harold and Burdsall 1965, 1968; Jack and Gaud 1997; Wei et al. 2010). Based on the results of our study, we conclude that the genus Picoa is a close relative of Geopora within the family Pyronemataceae. P. juniperi and P. lefebvrei, the two recognized species of the genus, 123 436 form together with Otidea subterranea (Smith and Healy 2009) the only known hypogeous and mycorrhizal truffles with a stereothecia in the family Pyronemataceae. Acknowledgments This work was partially supported by grants from the High Education and Scientific Research Ministry of Tunisia and the EU Project BIODESERT 245756 (FP7-Capacities-RegPot 2009-2). We thank Dr. Fatma MASMOUDI (CERT, Borj-Cedria) for helpful and assistance in the SEM observations. References Al-Scheikh M, Trappe JM (1983) Taxonomy of Phaeangiurn lefebvrei, a desert truffle eaten by birds. Can J Bot 61:1919–1925 Ammarellou A, Trappe JM (2007) A first Ascomycete genus (Picoa sp) record for the fungi flora of Iran. Pak J Biol Sci 10:1772 Ferdman Y, Aviram S, Roth-Bejerano N, Trappe JM, KaganZur V (2005) Phylogenetic studies of Terfezia pfeilii and Choiromyces echinulatus (Pezizales) support new genera for southern African truffles: Kalaharituber and Eremiomyces. Mycol Res 109:237–245 Fischer E (1897) Ascomyceten: Tuberaceen und Hemiasceen, Rabenhorst’s Kryptogamen—Flora von Deutschland, Oesterreich und der Schweiz, vol 1. 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