Tuber pulchrosporum Konstantinidis, Tsampazis, Slavova, Nakkas, Polemis, Fryssouli & Zervakis

Polemis, Elias, Konstantinidis, Georgios, Fryssouli, Vassiliki, Slavova, Monica, Tsampazis, Triantafyllos, Nakkas, Vasileios, Assyov, Boris, Kaounas, Vasileios & Zervakis, Georgios I., 2019, Tuberpulchrosporum sp. nov., a black truffle of the Aestivum clade (Tuberaceae, Pezizales) from the Balkan peninsula, MycoKeys 47, pp. 35-51 : 35

Tuber pulchrosporum Konstantinidis, Tsampazis, Slavova, Nakkas, Polemis, Fryssouli & Zervakis sp. nov. Fig. 1a

Type.

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!).

Diagnosis.

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.

Etymology.

" pulchrosporum " refers to the uniquely distinct/impressive ornamentation of the ascospores.

Description.

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. 1). Odour pleasant truffle-like.

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 1, 2).

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. 3); Q=1.0-2.2, Qm=1.43 ± 0.19; ornamentation with (0 –)1–2(– 4) thick veins across the long axis with few to several transverse outgrowths, rarely almost completely reticulate in maturity and then with (0 –)2–10(– 15) meshes in the longitudinal direction; circumferentially with 22-42 conical warts, with pointed or blunt, straight or curved apices, rarely forked, 1.5 –6(– 8) μm tall (Fig. 4); not reacting with Melzer’s reagent. Asci (64-) 78-96 (-121) × (50-) 65-84 (-98) μm (excluding stalk), globose, subglobose, ellipsoid, rarely subangular, with a short stalk, 6.5 –9(– 15) × 6.5 –7.5(– 10.5) μm, (1 –)2– 8-spored (Fig. 3).

Distribution and ecology.

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.

Additional collections examined (paratypes).

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).

Phylogenetic aspects.

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) ( Bonito et al. 2010a). ML and BI analyses yielded similar tree topologies and only the tree inferred from the Bayesian analysis is presented (Fig. 5). The morphologically variable genus Tuber is monophyletic (BS: 100%, PP: 1.00) and several lineages are revealed; for the purposes of this study, the following highly supported clades were included: Aestivum, Excavatum, Gennadii, Gibbosum, Latisporum, Maculatum, Macrosporum, Melanosporum, Puberulum, Regianum, Rufum, Tumericum (=Japonicum).

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.