Research Article |
Corresponding author: Georgios I. Zervakis ( zervakis@aua.gr ) Academic editor: Thorsten Lumbsch
© 2019 Elias Polemis, Georgios Konstantinidis, Vassiliki Fryssouli, Monica Slavova, Triantafyllos Tsampazis, Vasileios Nakas, Boris Assyov, Vasileios Kaounas, Georgios I. Zervakis.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Polemis E, Konstantinidis G, Fryssouli V, Slavova M, Tsampazis T, Nakkas V, Assyov B, Kaounas V, Zervakis GI (2019) Tuber pulchrosporum sp. nov., a black truffle of the Aestivum clade (Tuberaceae, Pezizales) from the Balkan peninsula. MycoKeys 47: 35-51. https://doi.org/10.3897/mycokeys.47.32085
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Knowledge on the diversity of hypogeous sequestrate ascomycetes is still limited in the Balkan Peninsula. A new species of truffle, Tuber pulchrosporum, is described from Greece and Bulgaria. Specimens were collected from habitats dominated by various oak species (i.e. Quercus ilex, Q. coccifera, Q. robur) and other angiosperms. They are morphologically characterised by subglobose, ovoid to irregularly lobed, yellowish-brown to dark brown ascomata, usually with a shallow basal cavity and surface with fissures and small, dense, almost flat, trihedral to polyhedral warts. Ascospores are ellipsoid to subfusiform, uniquely ornamented, crested to incompletely reticulate and are produced in (1–)2–8-spored asci. Hair-like, hyaline to light yellow hyphae protrude from the peridium surface. According to the outcome of ITS rDNA sequence analysis, this species forms a distinct well-supported group in the Aestivum clade, with T. panniferum being the closest phylogenetic taxon.
Ascomycota , Tuberaceae , truffle, ectomycorrhizal fungi, taxonomy, phylogeny, fungal diversity
The genus Tuber F.H. Wigg. (Ascomycota, Pezizales, Tuberaceae) is globally famous and historically appreciated for the production of hypogeous ascomata, known as ‘truffles’; several of them are highly prized due to their unique aroma and culinary value. Moreover, the genus is known for the symbiotic ectomycorrhizal associations that its members form with several gymnosperm and angiosperm forest-tree species as well as with orchids (
A continuous interest in the study of this particular group has resulted in several recent reports on new Tuber species from various parts of the world (e.g.
Although Tuber diversity is well documented in Europe (Bonito at al. 2010a,
In the frame of this work, several truffle specimens originating from north and central continental Greece and from Bulgaria were studied with respect to their morphology and phylogenetic relationships to other Tuber taxa and a new species is hereby proposed.
Specimens used for this study were collected during 2008–2017 from north and central Greece (Regions of Epirus, Thessaly, Eastern Macedonia and Thrace, Western Greece and Attica), as well as from Bulgaria (Regions of Eastern Stara Planina and Black Sea coast). Specimens are deposited in the fungaria of the Laboratory of General and Agricultural Microbiology (Agricultural University of Athens, ACAM), of the Institute of Biodiversity and Ecosystem Research (SOMF) and the authors’ personal collections. Macroscopic characters such as size, peridium surface texture, colour and odour were observed in fresh ascomata. Colour coding and terminology is derived from the “Flora of British Fungi – Colour Identification Chart” (
Microscopic characters were examined by hand-cut sections on fresh and dried material, using a Zeiss Axioimager A2 microscope under bright field and Differential Interference Contrast (DIC) and an AmScope T360B. Microphotographs were taken with the aid of a mounted digital camera (Axiocam). Microscopic observations were performed in water, 3% (w/v) potassium hydroxide (KOH) and Melzer’s reagent. To assess the ascospore size, a minimum of 30 mature ascospores from each type of asci (2 to 8-spored) were measured and dimensions are provided as (minimum) average ± standard deviation (maximum); quotient (Q), i.e. length divided by the width, was calculated for each ascospore and the median value (Qm) is given. For scanning electron microscopy (SEM), ascospores were scraped from the hymenial surface and mounted on aluminium foil, which was then fixed on a microscope holder and sputter-coated with gold. Observations were performed in JEOL JSM-5510.
Total genomic DNA was extracted from herbarium specimens using the Nucleospin Plant II DNA kit (Macherey and Nagel, Germany) following the manufacturer’s protocol with minor modifications. The internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) was amplified using the primer combination ITS1/ITS4 (
A total of 62 TuberITS rDNA sequences were used for phylogenetic analysis by including eight sequences of T. pulchrosporum sp. nov. and 54 sequences from GenBank (nine of them representing type specimens) which correspond to 31 Tuber taxa mainly of European distribution (Table
Details of ITS sequences deriving from Tuber pulchrosporum sp. nov. and from reference material used for the construction of the phylogenetic tree. Clades names are placed in the order they appear in Fig.
Species/ Clade | Collection code | GenBank Accession No. | Origin | Reference |
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Excavatum Clade | ||||
Tuber fulgens | M2435 | HM485358 | Italy |
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HMT37 | HM151976* | Austria | Urban et al. 2010 | |
Tuber excavatum | SA1TE | KJ524533* | Poland | Hilszczanska et al. 2014 |
JST62014 | KX354295 | Germany |
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Gennadii Clade | ||||
Tuber lacunosum | AH39255 | JN392212 | Spain |
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AH38932 | JN392213 | Spain |
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Tuber gennadii | B M1904 | HM485361 | Italy |
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AH39251 | JN392211 | Spain |
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AH31113 | JN392203 | Spain |
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AH38957 | JN392204 | Spain |
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Regianum Clade | ||||
Tuber bernardinii | 2172 | KY420104 | Italy | Merenyi et al. 2017 |
NA | KY420105 | Italy | Merenyi et al. 2017 | |
Tuber magentipunctatum | MO793 | KY420089 | Italy | Merenyi et al. 2017 |
ZB4293 | JQ288909** | Hungary | Merenyi et al. 2017 | |
Tuber regianum | ZB3081 | KY420098 | Slovakia | Merenyi et al. 2017 |
erd-2590 | KY420102 | Spain | Merenyi et al. 2017 | |
Macrosporum Clade | ||||
Tuber macrosporum | Macro1 | AF106885* | Italy | Rubini et al. 1998 |
HMSFI_TUBMAC/141207A | FM205634* | Slovenia | Grebenc et al. 2008 | |
Aestivum Clade | ||||
Tuber magnatum | JT19460 | HM485374 | Italy |
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GB12 | JQ925645 | Italy |
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Tuber malenconii | MA:Fungi:28384/ 02MLC | FM205597* | Spain | Grebenc et al. 2008 |
17110 | JF908743 | Italy |
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Tuber sinoaestivum | L4213 | KY081688* | Wang and Wang 2016 | |
JP-Zhang-140 | JN896355 | China |
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Tuber aestivum | TaeW016I-E134 | AJ888090 | Italy |
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S19 | HQ706002 | Slovakia |
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Tuber uncinatum | MA: Fungi: 24605 | FM205618* | Spain | Grebenc et al. 2008 |
228 | AJ492199 | Italy |
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Tuber mesentericum | CW105 | HM485375 | Sweden |
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UASWS1612 | KY197989* | Switzerland | Cochard et al. 2016 | |
Tuber panniferum | – | AF132507 |
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JT12835 | HM485380 | Spain |
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Tuber pulchrosporum sp. nov. | 1945 F8517 | MK113981 | Bulgaria | This work |
1961 F0388 | MK113982 | Bulgaria | This work | |
VN091 (holotype) | MK113975 | Greece | This work | |
GK3801 | MK113979 | Greece | This work | |
LT1183 | MK113976 | Greece | This work | |
GK9408 | MK113977 | Greece | This work | |
VK4482 | MK113980 | Greece | This work | |
GK6538 | MK113978 | Greece | This work | |
Multimaculatum Clade | ||||
Tuber multimaculatum | OSC 62169 | HM485377 | Spain |
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Rufum Clade | ||||
Tuber rufum | 1785 | EF362475 | Italy |
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S90 | JF926123 | Germany |
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Melanosporum Clade | ||||
Tuber pseudoexcavatum | T14_HKAS44325b | GU979039 | China |
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Tpse-yn05 | DQ329374 | China |
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Tuber regimontanum | ITCV 909 | EU375838 | Mexico |
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Tuber indicum | Ascocarpe I1 | AF300822 | China |
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HKAS 39501 | AY514305 | China |
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Tuber melanosporum | SB2-6 | MF693845 | France |
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P_Qr | KP972070 | Canada |
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Tumericum Clade | ||||
Tuber turmericum | BJTC FAN475 | KT758839 | China |
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BJTC FAN473 | KT758837 | China |
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Gibbosum Clade | ||||
Tuber oregonense | DUKE GB284 | FJ809874 | USA |
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Tuber gibbosum | OSC 40964 | FJ809863 | USA |
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Maculatum Clade | ||||
Tuber maculatum | A15 | AM406673 | Italy |
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Db-A | MH040280* | Sikora 2018 | ||
Latisporum Clade | ||||
Tuber latisporum | HKAS 44315 | DQ898183 | China |
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Tuber pseudosphaerosporum | BJTC Fan250 | KF744063 | China |
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Puberulum Clade | ||||
Tuber cistophilum | AH 39275 | JN392231 | Spain |
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Tuber borchii | Tar042 | KT165326 | Italy |
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Tuber sphaerospermum | AH38930 | JN392244 | Morocco |
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AH39190 | JN392246 | Spain |
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Outgroup | ||||
Choiromyces alveolatus | 22830 | AF501258 |
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p612i | EU697268* | Gordon 2008 |
Phylogenetic relationships of taxa were inferred by using maximum likelihood (ML) and Bayesian Inference (BI) through the CIPRES portal (www.phylo.org; Miller et al. 2010). ML analysis of the ITS dataset was conducted by RAxML v8.2 (
GREECE. Ioannina Prefecture: Ioannina city, 39°36'39"N, 20°50'05"E, 500 m alt., in soil under a pure stand of Quercus coccifera L., 27 Apr 2016, coll. V. Nakkas, VN091, holotype: ACAM 2016-007 (ACAM!); isotype: SOMF 29980 (SOMF!).
Ascomata 0.6–7(–10) cm in diam., subglobose, ovoid to irregularly lobed, usually with shallow basal cavity, surface with fissures and small, dense, almost flat trihedral to polyhedral warts, yellowish-brown to dark brown. Ascospores 25.0–37.0 × 18.2–25.6 μm in (1–)2–8-spored asci, ellipsoid to subfusiform on average, Qm=1.4, crested to incompletely reticulate. Hair-like, hyaline to light yellow-brown hyphae protruding from peridium surface.
T. panniferum, the closest phylogenetically-related species, produces smaller ascospores (23–26 × 18–20 μm), broadly ellipsoid to subglobose on average, with isolated warts; moreover, the peridium surface is woolly-felted due to the presence of dense rusty brown hair-like hyphae.
“pulchrosporum” refers to the uniquely distinct/impressive ornamentation of the ascospores.
Ascomata 0.6–7(–10) cm in diameter, tuberous, subglobose, ovoid to irregularly lobed, usually depressed with a shallow - occasionally prominent - basal cavity (excavated), covered up with whitish to yellowish rhizomorphs, fragile, initially greyish to yellowish-brown [fawn (29), sienna (11), fulvous (12)], darkening in maturity to brown [snuff brown (17), umber (18), bay (19), to date brown (24)] or with some shades of purple tinges [purplish date (22), purplish chestnut (21) to brown vinaceous (25)], sometimes with darker black [fuscous black (38)] spots, surface rarely almost smooth, usually rough, with fissures and small, dense, almost flat trihedral to polyhedral warts. Gleba with one of more cavities, initially pinkish-grey [vinaceous buff (31), clay pink (30)], then greyish-brown [milky coffee (28)], yellowish-brown [fulvous (12)], brown [snuff brown (17), umber (18), bay (19)], to purplish-brown in maturity [purplish date (22) to purplish chestnut (21)], with bay (19) to rusty tawny (14) coloured areas close to the cavity, marbled with relatively few and thick white veins, that sometimes are reddening (Fig.
Peridium 120–370 μm thick, consisting of two layers; the outer layer 50–160 μm thick, pseudoparenchymatous, composed of yellowish-brown and subglobose inwards to subangular dark brown cells outwards; 4.0–16.3 × 2.5–13.2 μm, thick-walled (1.5–2.5 μm); the inner layer 70–210 µm, composed of pale yellow or hyaline and thick-walled, interwoven hyphae, 2–10 μm in diameter, forming an intricate texture, becoming agglutinated when dried. Surface with abundant isolated, hyaline to golden-yellow (in water or KOH), thick-walled hair-like hyphae (walls 1.0–1.5 μm), 30–140 μm long (occasionally exceeding 300 μm in Bulgarian specimens) and 2.5–4.5 μm broad at base, 1–2 septate (Figs
Ascospores hyaline when young then yellowish, yellow-brown to brown, at most ellipsoid to subfusiform, some broadly ellipsoid, subglobose to globose, rarely almost limoniform in initial stages, thin-walled and smooth when young, becoming thick-walled at maturity, walls 2–3.5(–4) μm thick, usually crested to incompletely reticulate, measured (excluding the ornamentation) in the rare 1-spored asci (28–) 46.7±7.4 (–57) × (20–) 29.4±4.6 (–34) μm, in 2-spored asci (27–) 39.5±5.8 (–53) × (21–) 27.3±4.2 (–41) μm, in 3-spored asci (24–) 34.5±5.3 (–49) × (19–) 24.5±2.6 (–31) μm, in 4-spored (21–) 30.9±4.9 (–39) × (18–) 22.2±2.7 (–30) μm, in 5-spored asci (22–) 30.3±3.7 (–44) × (16–) 21.2±2.2 (–28) μm, in 6-spored asci (22–) 28.9±4.6 (–37) × (17–) 20.6±2.0 (–28) μm, in 7-spored asci (21–) 27.8±3.3 (–35) × (13–) 19.9±2.7 (–27) μm and in 8-spored asci (20–) 25.4±2.6 (–31) × (14–) 18.4±3.1 (–26) μm (Fig.
Hypogeous, in soil, appearing solitary or in small groups from March to June, under Quercus sp., Q. coccifera or Q. ilex L. or under Carpinus sp. or in mixed stands of Quercus sp. and Pinus nigra J.F. Arnold or of Q. ilex and Pinus halepensis Miller or of Quercus robur L., Corylus sp., Carpinus sp. and Acer sp. It seems to be rather common in continental (northern and central) Greece, while it also occurs in the regions of Eastern Stara Planina and the Black Sea coast of Bulgaria.
GREECE. Xanthi Prefecture: Toxotes, in soil under a mixed stand dominated by Q. coccifera, 20 June 2008, GK3186b (ACAM 2010-127), coll. P. Panagiotidis. Aitoloakarnania Prefecture: Xiromero, in soil under pure forest of Quercus sp., 10 May 2009, GK3801 (ACAM 2010-129), coll. Ch. Chrysopoulos and K. Giatra (GenBank: MK113979); Xiromero, in soil under pure forest of Quercus sp., 10 May 2009, GK3799 (ACAM 2010-128), coll. Ch. Chrysopoulos and K. Giatra. Trikala Prefecture: Koziakas Mt., in soil under mixed forest of Quercus sp. and P. nigra, 2 April 2013, GK6538 (ACAM 2013-073), coll. K. Papadimitriou (GenBank: MK113978); Koziakas Mt., in soil under mixed forest of Quercus sp. and P. nigra, 2 April 2013, GK6537 (ACAM 2013-074), coll. K. Papadimitriou. Ioannina Prefecture: Metsovo, in soil under pure stand of Q. coccifera, 18 April 2016, GK9408 (ACAM 2016-001), coll. A. Bideris (GenBank: MK113977); Metsovo, in soil under pure stand of Q. coccifera, 19 April 2016, GK9409 (ACAM 2016-002), coll. A. Bideris; Metsovo, in soil under pure stand of Q. coccifera, 19 April 2016, GK9410 (ACAM 2016-003), coll. A. Bideris; Demati, in soil under pure stand of Q. coccifera, 22 March 2017, GK10231 (ACAM 2017-033), coll. A. Bideris. Attica Prefecture: Katsimidi, in soil under mixed forest of Q. ilex and P. halepensis, 22 March 2016, VK4482 (ACAM 2016-004), coll. V. Kaounas (GenBank: MK113980); Katsimidi, in soil under mixed forest of Q. ilex and P. halepensis, 12 April 2016, VK4506 (ACAM 2016-005), coll. V. Kaounas (GenBank: MK113980). Ioannina Prefecture: Neochoropoulo, in soil under a mixed stand of Q. coccifera and Q. ilex, 27 April 2016, LT1183 (ACAM 2016-006), coll. V. Nakkas (GenBank: MK113976). BULGARIA. Varna, Dolishte village, in soil under pure stand of Carpinus sp., 07 June 2017, MSL 1945 F8517 (SOMF 29978; ACAM 2017-034), coll. R. Radev (GenBank: MK113981). Sliven, in soil under a mixed stand of Quercus robur, Corylus sp., Carpinus sp. and Acer sp., 09 August 2017, MSL 1961 F0388 (SOMF 29979; ACAM 2017-035), coll. K. Pilasheva & P. Neikov (GenBank: MK113982).
The resultant ITS sequence data comprises of 64 sequences which were aligned at 780 sites, 738 of which represent the ITS1-5.8S-ITS2 region, i.e. between the end of the SSU motif (CATTA) and the beginning of LSU motif (TAGGG) (
Phylogenetic tree inferred from Bayesian analysis including 62 ITS sequences assigned to 31 Tuber taxa, including members of major clades of the genus. Sequences are labelled with Latin binomials, GenBank accession numbers and geographic origin. T. pulchrosporum sp. nov. is indicated in boldface. Reference sequences deriving from type material are underlined. Choiromyces alveolatus (Tuberaceae) was used as the outgroup. Bootstrap (BS) values from Maximum Likelihood (ML) analysis (≥ 70%) and Posterior Probabilities (PPs) from Bayesian Inference (≥ 0.95) are shown at the nodes of branches.
According to the phylogenetic analysis performed, T. pulchrosporum belongs to the Aestivum clade. All eight sequences of this new taxon form a distinct highly supported subclade (BS: 100%, PP: 1.00). Greek specimens possessed almost identical ITS sequences (99.8 – 100%) and so did Bulgarian samples, whereas the comparison between collections from the two countries resulted in sequence identity values of 98.13 ± 0.08%. In total, intraspecific sequence identity values for T. pulchrosporum exceeded 98% (i.e. 98.05 – 100%). The new species is sister to T. panniferum (BS: 100%, PP: 1.00); the respective sequences demonstrated low sequence identity (73.21 – 75.08%) further evidencing their distinct taxonomic status.
The molecular analysis evidenced that the eight sequences representing T. pulchrosporum are grouped within the Aestivum clade by forming a distinct terminal group supported with high BS and PP values. The closest phylogenetic relative of T. pulchrosporum is T. panniferum Tul. & C. Tul., i.e. a Mediterranean species with analogous ecological preferences (
By morphology alone, T. pulchrosporum is easily distinguishable within the Aestivum clade since no other species produces ascospores bearing such a uniquely crested ornamentation. The more distant T. aestivum (Wulfen) Spreng. (including T. uncinatum Chatin) and T. sinoaestivum J.P. Zhang & P.G. Liu could be distinguished macroscopically thanks to their blackish peridial surface with prominent pyramidal warts and ascospores bearing a complete reticulum. Ascospores of T. mesentericum Vittad. show some affinity in their outline to those of T. pulchrosporum but they clearly possess a much more reticulate network; moreover, the peridial surface is black with pyramidal warts as in T. aestivum.
Although phylogenetically more distant, some other species with asci containing 1–8 ascospores may superficially resemble T. pulchrosporum. Hence, T. regianum Montecchi & Lazzari, the recently described T. magentipunctatum Z. Merényi, I. Nagy, Stielow & Bratek and T. bernardinii Gori, all belonging to the Regianum clade (
This work was partly financed by the IBER-BAS project “Taxonomy, conservation and sustainable use of fungi” and by research funding provided to GZ. The following individuals are acknowledged for kindly providing truffle specimens for examination: A. Aggelopoulou, A. Bideris, Ch. Chrysopoulos, K. Giatra, P. Neikov, P. Panagiotidis, K. Papadimitiriou, K. Pilasheva and R. Radev.