The Diversity of the Genus Tuber in Greece—A New Species to Science in the Maculatum Clade and Seven First National Records

Abstract

Ectomycorrhizal fungi of the genus Tuber (Ascomycota) produce hypogeous ascomata commonly known as truffles. Despite their high ecological and economic importance, a considerable gap of knowledge exists concerning the diversity of Tuber species in the eastern Mediterranean region. In the frame of this study, more than 200 Tuber collections, originating from various regions of Greece, were examined. A new species to science, i.e., Tuber leptodermum, is formally described. Tuber leptodermum is grouped in the Maculatum clade, as revealed by the ITS and LSU rDNA concatenated phylogenetic tree, and appears as sister to T. foetidum. In addition, T. leptodermum exhibits distinct morphoanatomic features: it produces medium-sized, dark-brown ascomata with a thin pseudoparenchymatous peridium, composed of globose-to-angular cells and forms one-to-four-spored asci containing reticulate–alveolate, ellipsoid ascospores with broad meshes. Thirty other phylogenetic species are identified: seven of them (i.e., T. anniae, T. buendiae, T. conchae, T. dryophilum, T. monosporum, T. regianum and T. zambonelliae) constitute new records for the Greek mycobiota, while the presence of five other species is molecularly confirmed for the first time. Moreover, the existence of ten undescribed phylogenetic species is revealed, six of which are reported for the first time in Greece. Several taxonomic and phylogenetic issues and discrepancies in the genus Tuber are discussed in relation to the new findings.

1. Introduction

The term “true truffles” is used to describe the subterranean spherical and enclosed ascomata produced by fungi of the genus Tuber P.Micheli ex F.H.Wigg. (Pezizales, Ascomycota), which includes several gastronomically valuable and highly prized species [1,2,3]. These fungi form obligate ectomycorrhizal (ECM) relationships with various plant genera, which link the presence of truffles to that of their plant symbionts, depending on species-specific and environmental factors [4]. In addition, since truffle spores are mainly dispersed through the consumption of ascocarps by mycophagous animals, their distribution range is spatially limited [1,5]. During the last glacial periods, survival refugia for various species were established in favorable areas of southern Europe (e.g., in the Balkan peninsula); these refugia became the cradle for species recolonization of the continent [6]. Truffles follow a similar pattern of distribution, which is also affected by their unique biology and past biogeographic incidents [4,7,8,9].

The genus Tuber includes approx. 200 species which are grouped into 13 major phylogenetic clades, i.e., Aestivum, Excavatum, Gennadii, Gibbosum, Latisporum, Macrosporum, Maculatum, Melanosporum, Multimaculatum, Puberulum, Regianum, Rufum and Tumericum-Japonicum [1,10,11]. Several studies have dealt with the diversity of Tuber species in Europe; however, some regions—like the southern Balkans—still remain relatively understudied [2,12,13,14].

Regarding Greece in particular, the earliest documented reference of a “truffle” species can be found in the monumental work Flora Graeca [15], where “Tuber album” is mentioned; this finding, however, corresponds to Terfezia arenaria (Moris) Trappe, according to Chatin [16]. Xavier Laderer [17], a pharmacist at the palace during the reign of King Otto, reported the existence of Tuber cibarium. In addition, Chatin [16] described Terfezia gennadii based on collections made by Panagiotis Gennadios in the Peloponnese; this species was subsequently transferred to the genus Tuber, as T. gennadii (Chatin) Pat. Later, the first record of Tuber aestivum Vittad. was included in the Fungus-host index for Greece [18].

Hence, until the end of the 20th century, the diversity of the genus Tuber in Greece remained largely unknown, as reflected in the first national checklist of larger Ascomycota [19], which included the aforementioned records only. However, in recent years, the collection of truffles has become popular, while pioneering members of citizen science associations (e.g., the Greek Mushroom Society, GMS) have started identifying specimens using traditional taxonomy. A significant change in the knowledge of the genus Tuber in Greece is apparent from 2007 onwards, as reflected by the publication of a species checklist [20] and mushroom field guides [21,22,23,24,25]. The first study using DNA sequencing dealt with truffle specimens identified as T. borchii Vittad., T. gennadii, T. oligospermum (Tul. & C. Tul.) Trappe and T. puberulum Berk. & Broome [26], and was followed by reports of the presence of T. brumale Vittad. haplogroup II [8], T. magnatum Picco [9], T. mesentericum Vittad., T. bituminatum Berk. & Broome, T. suave Pacioni & M. Leonardi [27] and T. magentipunctatum Z. Merényi, I. Nagy, Stielow & Bratek [28]. In addition to these findings, two new species to science found in Greece have recently described, i.e., T. pulchrosporum Konstantinidis, Tsampazis, Slavova, Nakkas, Polemis, Fryssouli & Zervakis from material collected in spring to early summer in Bulgaria and continental Greece [29], and T. aereum Polemis, Daskalopoulos and Zervakis from specimens found in autumn and winter in Naxos Island [30]. At present, 26 species (including 4 phylogenetic species yet to be formally described) of this genus are known to exist in Greece, while the presence of only 15 of them is supported by molecular approaches.

The objective of the present work was to assess the diversity and distribution of the genus Tuber in Greece by examining numerous collections from a large range of geographic areas and a variety of habitats, through the combined use of morphoanatomical characters, ecological features and phylogenetics.

2. Materials and Methods

2.1. Specimens Studied

The biological material examined in the frame of this study was derived from the following sources: (a) from dried specimens previously deposited in the fungarium of the Laboratory of General and Agricultural Microbiology, Agricultural University of Athens (ACAM), or in personal collections of the GMS’s members [G. Konstantinidis (GK), V. Kaounas (VK), N. Tsilis (NT) and M. Gkilas (MG)]; and (b) from fresh ascomata sampled either by the authors or by collaborating truffle collectors. All the fresh samples received were subsequently dried and stored in the fungarium ACAM. In total, 242 collections (several of them including more than one specimen) were identified through the combined use of morphoanatomic criteria and DNA sequencing, while 63 of them were further subjected to phylogenetic analysis (Table 1; Supplementary Material, Table S1).

2.2. Morphoanatomic Features

Each sample, fresh or dried, was macroscopically examined and photographed using a Nikon SMZ18 stereoscope (Nikon Corporation, Tokyo, Japan) with the OCULAR vers. 2.0 software (Teledyne Photometrics, Tucson, AZ, USA). Color description was based on the “Flora of British Fungi: Colour Identification Chart” [31], and the respective codes are provided in braces. Microscopic observations were performed in water and in 3–5% (w/v) potassium hydroxide (KOH). Microanatomical features of the ascomata were examined using an Olympus BX53F2 (Olympus Corporation, Tokyo, Japan) or a Zeiss AxioImager A2 (Carl Zeiss Microscopy, Oberkochen, Germany) microscope, and were photographed with either an Olympus DP74 camera using the cellSens Entry software (Olympus Life Science, Waltham, MA, USA), or with an Axiocam 305 color camera using the ZEN vers. 2.3 lite software (Carl Zeiss Microscopy, Oberkochen, Germany), respectively. Photographs were taken under bright field, phase contrast and/or differential interference contrast (DIC) microscopy.

Initial evaluation of Tuber collections was based on their morphoanatomic and ecological features, in accordance with taxonomic keys included in various publications [27,28,32], and with a monograph on European Tuber species [12]. For selected specimens (e.g., those representing new species to science, and those corresponding to the first national records) the following microscopic features were thoroughly examined: the peridium width (total, and external if present), asci and stalk size, size of parenchymatic cells (if present), ascospore number per ascus, ascospore dimensions (per ascus category and in total), height of meshes or spines, and (in reticulate ascospores) number of meshes across the ascospore length. As a general rule, a minimum of 30 elements were measured for each feature by using Piximètre vers. 5.10 software (http://www.piximetre.fr/; France; accessed on 1 February 2025). Dimensions of various features are provided as follows: (minimum), range of length, (maximum), while the quotient (Q) value for ascospores is also provided, i.e., length divided by width. Average values (Av) were also calculated in the case of asci and ascospores; in addition, percentages are provided for the number of ascospores per ascus, and ranges are provided for the number of meshes along the spore length. Especially in the case of T. regianum Montecchi & Lazzari, the ascospore volume is also provided in accordance with Merenyi et al. [33] and Cseh et al. [28]: 0.523 × (width)² × length.

2.3. DNA Extraction, Amplification and Sequencing of Phylogenetically Informative Markers

The total genomic DNA was isolated from dried or fresh material as follows: samples were ground to fine powder in the presence of liquid nitrogen, mixed with warm (60 °C) DNA lysis buffer (1% CTAB, 10 mM Na₂EDTA, 0.7 M NaCl, and 50 mM Tris-HCl pH 8), and then extracted with phenol:chloroform:isoamyl alcohol (25:24:1), and precipitated with ethanol. The two regions of the nuclear ribosomal repeat unit, i.e., the internal transcribed spacer (ITS) and a fragment of the ribosomal large subunit gene (LSU), were amplified by the polymerase chain reaction (PCR) in a MiniAmp Plus Thermal Cycler (Applied Biosystems, CA, USA), using 0.8 μL of each primer (0.32 μM), 12.5 μL of FastGene^® Optima Polymerase hotstart ready mix (NIPPON Genetics Europe, Düren, Germany), 11.5 μL of UltraPure™ DNase/RNase-Free Distilled Water (Thermo Fisher Scientific, Waltham, MA, USA) and 1 μL of DNA template (ca. 10 ng μL⁻¹). The ITS was amplified through the use of ITS1 and the ITS1f/ITS4 primer pair [34,35] under the following conditions: initial denaturation at 95 °C for 4 min, followed by 35 cycles at 95 °C for 30 sec, annealing at 55 °C for 30 sec, extension at 72 °C for 1 min and final extension at 72 °C for 7 min. In addition, partial sequences of the LSU were amplified by the primer pair LROR/LR5 [36] under the following conditions: initial denaturation at 95 °C for 4 min, followed by 35 cycles at 94 °C for 1 min, annealing at 52 °C for 50 sec, extension at 72 °C for 1 min and final extension at 72 °C for 10 min. Electrophoresis was performed with 1% (w/v) agarose gel and purification of the PCR product was performed with the PureLink Quick PCR kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), and the purified products were quantified using the MULTISKAN SkyHigh Microplate Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Sequences were obtained by using the same forward and reverse primers (as before in the amplification procedure) in an automated ABI sequencer (Life Technologies, USA) at CeMIA SA (Greece). FinchTV vers. 1.4.0 software (Geospiza, Inc., Denver, CO, USA) was used for chromatogram visualization and inspection of sequencing quality. Consensus sequences were generated using the SeqMan module of Lasergene vers. 7.1 software (DNAStar, Madison, WI, USA), manually edited to remove or replace all ambiguous characters, and cross-checked against public databases, including the international nucleotide sequence database collaboration (INSDC) [37] and UNITE [38]. Validated sequences were submitted to GenBank, and the pertinent accession numbers appear in Table 1 and in Supplementary Table S1.

Table 1. Details of biological material from the genus Tuber used in the phylogenetic analyses performed in the frame of this study: the phylogenetic clade and taxon, collection code, geographic origin, GenBank accession no. for ITS and LSU sequences, and respective references. The material from which sequences were generated for the first time is presented in bold typeface, while material corresponding to type specimens is underlined.

	CLADE/Taxon	Collection Code	Origin	ITS	LSU	Reference
	AESTIVUM
1	T. aestivum	AQUI 10150	Italy	MZ423173		[32]
2	T. aestivum	S8	Slovakia	HQ285310		[39]
3	T. aestivum	2PL-M(u)	Poland	KX028767		[40]
4	T. aestivum	ACAMTub361	Greece	PP725740		[41]
5	T. aestivum	ACAMTub271	Greece	PP918676		This study
6	T. aestivum	ACAMTub376	Greece	PP918694		This study
7	T. bituminatum	AQUI 9833	Italy	OL711611		[27]
8	T. bituminatum	AQUI 8579	Greece	OL711613		[27]
9	T. bituminatum	ACAMTub310	Greece	PP918764		This study
10	T. bituminatum	ACAMTub421	Greece	PP918781		This study
11	T. magnatum	TO HG3458	Italy	MZ423175		[32]
12	T. magnatum	HHD19491	Turkey	PP239641		Unpublished
13	T. magnatum	B1	Italy	AJ586268		[42]
14	T. magnatum	ACAMTub055	Greece	PP918868		This study
15	T. magnatum	ACAMTub156	Greece	PP918870		This study
16	T. mesentericum	AQUI 9717	Italy	OL711593		[27]
17	T. mesentericum	AQUI 10238	France	OL711600		[27]
18	T. mesentericum	AQUI 10230	Greece	OL711607		[27]
19	T. mesentericum	ACAMTub111	Greece	PP918880		This study
20	T. mesentericum	ACAMTub250	Greece	PP918885		This study
21	T. panniferum	JT12835	Spain	HM485380		[43]
22	T. panniferum	MUB:Fung-0973	Spain	MN962725		[44]
23	T. panniferum	ACAMTub269	Greece	PP918896		This study
24	T. panniferum	ACAMTub385	Greece	PP918897		This study
25	T. pulchrosporum	VN091	Greece	MK113975		[29]
26	T. pulchrosporum	1961 F0388	Bulgaria	MK113982		[29]
27	T. suave	AQUI 7131	Italy	OL711623		[27]
28	T. suave	AQUI 10229	Greece	OL711627		[27]
	EXCAVATUM
29	T. excavatum	AP-T63a	Slovenia	FM205555		Unpublished
30	T. excavatum	Tub10	Italy	HM152012		Unpublished
31	T. aff. excavatum 1	ACAMTub151	Greece	PP918825		This study
32	T. aff. excavatum 1	ACAMTub162	Greece	PP918826		This study
33	T. excavatum	SFI:TUBEXC/251008B	Slovenia	FN433141		Unpublished
34	T. fulgens	AP-T60	Slovenia	FM205568		Unpublished
35	T. excavatum	zb3377	Hungary	HM152023		Unpublished
36	T. excavatum	AP-T11	Slovenia	FM205554		Unpublished
37	T. aff. excavatum 2	ACAMTub028	Greece	PP918828		This study
38	T. aff. excavatum 2	ACAMTub316	Greece	PP918829		This study
39	T. excavatum	SFI:TUBEXC/121008	Slovenia	FN433142		Unpublished
40	T. excavatum	SA1TE	Poland	KJ524533		Unpublished
41	T. aff. excavatum 3	ACAMTub168	Greece	PP918841		This study
42	T. aff. excavatum 3	ACAMTub320a	Greece	PP918851		This study
43	T. excavatum	BM100	Spain	FJ748899		[45]
44	T. excavatum	MA:Fungi:54695	Spain	FM205561		Unpublished
45	T. fulgens	zb3335_205	Hungary	HM152021		Unpublished
46	T. fulgens	CMI-Unibo_5034	Iran	MW884551		[46]
47	T. fulgens	HMT44	Italy	HM151979		Unpublished
48	T. fulgens	ACAMTub170	Greece	PP918853		This study
49	T. fulgens	ACAMTub312	Greece	PP918854		This study
50	T. iranicum	CMI-Unibo 4939	Iran	MN854634		[47]
51	T. iranicum	CMI-Unibo 4965	Iran	MN854635		[47]
	GENNADII
52	T. gennadii	B M1904	Italy	HM485361		[43]
53	T. gennadii	AH39251	Spain	JN392211		[26]
54	T. aff. gennadii	ACAMTub050	Greece	PP918857		This study
55	T. aff. gennadii	ACAMTub131	Greece	PP918858		This study
56	T. gennadii	AH38957	Spain	JN392204		[26]
57	T. gennadii	VK2141	Greece	JN392205		[26]
58	T. gennadii	AH38955	Spain	JN392207		[26]
59	T. gennadii	MRG496	Spain	OQ565278		[48]
60	T. conchae	MRG486	Spain	OQ565276		[48]
61	T. conchae	ACAMTub046	Greece	PP918816		This study
62	T. lacunosum	AH38912	Morocco	JN392214		[26]
63	T. lacunosum	MRG692	Spain	OQ565280		[48]
64	T. lucentum	j957	Spain	MN437527		[49]
65	T. lucentum	MUB:Fung-j825	Spain	NR184909		[49]
	MACROSPORUM
66	T. calosporum	HKAS:88790	China	KT444598		[50]
67	T. calosporum	HKAS:88751	China	KT444600		[50]
68	T. glabrum	BJTCFan228	China	KF002731		[51]
69	T. glabrum	BJTCFan232	China	KF002727		[51]
70	T. macrosporum	CMI-Unibo_4937	Iran	MW884553		[46]
71	T. macrosporum	CMI-Unibo_4938	Iran	MW884554		[46]
72	T. macrosporum	FHS-449	Serbia	FM205664		[14]
73	T. macrosporum	IC28092233	Spain	PP948901		Unpublished
74	T. macrosporum	ITA_014s	Italy	KP738351		[52]
75	T. macrosporum	ACAMTub052	Greece	PP918859		This study
76	T. macrosporum	ACAMTub110	Greece	PP918860		This study
77	T. macrosporum	ACAMTub252	Greece	PP918862		This study
78	T. macrosporum	ITA_003V	Italy	KP738380		[52]
79	T. macrosporum	ITA_008	Italy	KP738388		[52]
80	T. macrosporum	FRA_002	France	KP738361		[52]
81	T. monosporum	ACAMTub455	Greece	PP918907	PP892729	This study
82	T. sinomonosporum	BJTCFan150	China	KF002729		[51]
	MACULATUM
83	T. arnoldianum	RH1619	USA	KU186913		[53]
84	T. arnoldianum	FLAS:F-71902	USA	OR762689	PP337424	Unpublished
85	T. arnoldianum	FLAS:F-71880	USA	OR762672	PP337406	Unpublished
86	T. arnoldianum	AMC105	USA	OP413000		Unpublished
87	T. aztecorum	ITCV 993 ITCV	Mexico	NR159044		[54]
88	T. aztecorum	GG993 FLAS	Mexico	KY271791		[54]
89	T. beyerlei	JT32597	USA	HM485408		[43]
90	T. beyerlei	Berch 0042	Canada	KP972075		[55]
91	T. brennemanii	MES653	USA	MF611779	KY565252	[56]
92	T. brennemanii	RH1746	USA	MF611791	KY565258	[56]
93	T. excelsum-reticulatum	BJTC FAN863	China	OM265272	OM366224	[11]
94	T. excelsum-reticulatum	BJTC FAN758	China	OM265264	OM366218	[11]
95	T. foetidum	ZB2489	Hungary	JQ288906	JF261362	Unpublished
96	T. foetidum	ZB2452	Hungary	JQ288905	JF261361	Unpublished
97	T. foetidum	JCAS0140001000096	Spain	MH703905		[57]
98	T. foetidum	ZB3454	Finland	FN568055		[58]
99	T. foetidum	B-2489	Hungary	AJ557544		[13]
100	T. foetidum	B-2452	Hungary	AJ557543		[13]
101	T. leptodermum	ACAMTub475	Greece	PQ877422	PQ881954	This study
102	T. leptodermum	ACAMTub359	Greece	PP918908	PP905688	This study
103	T. leptodermum	ACAMTub379	Greece	PP918909	PP905689	This study
104	T. leptodermum	ACAMTub380	Greece	PP918910	PP905690	This study
105	T. leptodermum	ACAMTub382	Greece	PP918911	PP905691	This study
106	T. hubeiense	HMAS 60233	China	KT067688	KT067694	[11]
107	T. lusitanicum	MUB:Fung-0986	Spain	MT621651	MT705332	[59]
108	T. lusitanicum	MUB:Fung-0988	Spain	MT621653		[59]
109	T. lusitanicum	MUB:Fung-0990	Spain	MT621655		[59]
110	T. maculatum		Italy	AF003919		Unpublished
111	T. maculatum	herbarium 1967	Italy	EU753269		Unpublished
112	T. maculatum	Imatra_3	Finland	MZ389968		[60]
113	T. maculatum	FLAB22	Uruguay	PP297668		[61]
114	T. maculatum	BJTC FAN876	China	OM265278	OM366228	[11]
115	T. maculatum	BJTC FAN868	China	OM265274	OM366227	[11]
116	T. miquihuanense	ITCV885	Mexico	HM485414	JF419292	[43]; [62]
117	T. pseudomagnatum	BJTC FAN163	China	JQ771192		[63]
118	T. pseudomagnatum	BJTC FAN299	China	OM265244	OM366195	[11]
119	T. pseudomagnatum	BJTC FAN392	China	KP276184	KP276193	[64]
120	T. pseudomagnatum	BJTC FAN390	China	OM265243		[11]
121	T. pseudomagnatum	BJTC FAN389	China	OM265242		[11]
122	T. rapaeodorum	BG Kew K(M)128884	England	EU784429		[65]
123	T. rapaeodorum	CMI-UNIBO 2483	Armenia	DQ011849		Unpublished
124	T. walkeri	RH5211	USA	JF419260		[62]
125	T. walkeri	FLAS:MES-274	USA	MT156441		Unpublished
126	T. walkeri	RH794	USA	JF419258	JF419308	[62]
127	T. whetstonense	JT25783	USA	HM485392	JF419304	[43]; [62]
128	T. whetstonense	SOC 756 OSC 111412	USA	AY830855		[66]
129	T. whetstonense	FLAS:MES-210	USA	MT156439		Unpublished
130	T. wumengense	BJTC FAN218A	China	KT067682	KT067707	[64]
131	T. wumengense	HMAS 60229	China	KT067687		[64]
132	T. wumengense	BJTC FAN586	China	OM265254		[11]
	MELANOSPORUM
133	T. brumale	G1126	Greece	KF550998		[8]
134	T. brumale	BTb2p2	Turkey	KF551063		[8]
135	T. brumale	FHS-460	Serbia	FM205660		[14]
136	T. brumale	CMI-Unibo_5001	Iran	MW829419		[46]
137	T. brumale	MA:Fungi:28373	Spain	FM205550		[14]
138	T. brumale	TB59k1	France	KF550974		[8]
139	T. brumale	ACAMTub159	Greece	OP850807		This study
140	T. brumale	ACAMTub233	Greece	PP918804		This study
141	T. brumale	ACAMTub456	Greece	PP918814		This study
142	T. cryptobrumale	HNHM<HUN>:BP107922	Hungary	KU203777		[33]
143	T. cryptobrumale	HNHM<HUN>:BP107923	Hungary	KU203778		[33]
144	T. indicum	Tind-gs01	China	DQ375490		[67]
145	T. indicum	Tind-gs05	China	DQ375491		[67]
146	T. melanosporum	AQUI 10152	Italy	MZ423176		[32]
147	T. melanosporum	MEL178	France	EU555385		[68]
148	T. melanosporum	ACAMTub256	Greece	PP918878		This study
149	T. petrophilum	LJU-GIS-TUBsp_xx1010A	Serbia	HG810883		[69]
150	T. petrophilum	LJU-GIS-TUBsp_xx1010D	Serbia	HG810886		[69]
151	T. thracicum	SOMF:30882	Bulgaria	OR666020		[70]
152	T. thracicum	SOMF:30883	Bulgaria	OR666027		[70]
	PUBERULUM
153	T. oligospermum	CMI-UNIBO 4230	Morocco	KF021624		[71]
154	T. oligospermum	CMI-UNIBO 4234	Morocco	KF021622		[71]
155	T. oligospermum	AH39002	Italy	JN392248		[26]
156	T. oligospermum	AH37861	Italy	JN392251		[26]
157	T. oligospermum	GK3060	Greece	JN392249		[26]
158	T. aff. oligospermum 1	ACAMTub075	Greece	PP918887		This study
159	T. oligospermum	MUB:Fung-1049	Spain	PQ281432		Unpublished
160	T. oligospermum	MUB:Fung-1050	Israel	PQ281433		Unpublished
161	T. oligospermum	GK5388	Greece	JN392263		[26]
162	T. aff. oligospermum 2	ACAMTub062	Greece	PP918889		This study
163	T. oligospermum	HAI-D-102	Israel	JN392271		[26]
164	T. oligospermum	VK2042	Greece	JN392254		[26]
165	Soil sample	S052	Iran	UDB0765470		Unpublished
166	T. aff. oligospermum 3	ACAMTub133	Greece	PP918893		This study
167	T. anniae	JT13209	USA	HM485338		[43]
168	T. anniae	Juva_1	Finland	MZ389970		[60]
169	T. anniae	ACAMTub024	Greece	PP918746		This study
170	T. borchii	AP-T30	Slovenia	FM205497		[14]
171	T. borchii	CMI-Unibo_5047	Iran	MW829422		[46]
172	T. borchii	17Bo	Italy	DQ679802		[72]
173	T. borchii	ACAMTub135	Greece	PP918787		This study
174	T. borchii	ACAMTub334	Greece	PP918792		This study
175	T. borchii	ACAMTub371A	Greece	PP918796		This study
176	T. borchii	AQUI 10151	Italy	MZ423174		[32]
177	T. borchii	GK5597	Greece	JN392228		[26]
178	T. borchii	CMI-UNIBO 3026	Italy	FJ554493		[72]
179	T. borchii	MUB:Fung-1036	Spain	PQ273773		Unpublished
180	T. borchii	ACAMTub120A	Greece	PP918786		This study
181	T. borchii	ACAMTub207	Greece	PP918802		This study
182	T. borchii	ACAMTub377	Greece	PP918803		This study
183	T. dryophilum		Italy	AF003917		Unpublished
184	T. puberulum	GK4352	Greece	JN392223		[26]
185	T. dryophilum	GB69	Italy	HM485353		[10]
186	T. puberulum	GK5601	Greece	JN392224		[26]
187	T. dryophillum	ACAMTub043	Greece	PP918819		This study
188	T. dryophillum	ACAMTub134	Greece	PP918821		This study
189	T. dryophillum	ACAMTub182	Greece	PP918822		This study
190	T. dryophillum	ACAMTub204	Greece	PP918824		This study
191	T. lijiangense	BJTC FAN307	China	KP276188		[64]
192	T. cf. lijiangense	BJTC FAN148	China	OM286798		[11]
193	T. puberulum	Berk. & Broome TL11885	Denmark	AJ969626		[73]
194	T. puberulum	Berk. & Broome TL3857	Denmark	AJ969625		[73]
	REGIANUM
195	T. magentipunctatum	ZB4293	Hungary	JQ288909		[74]
196	T. magentipunctatum	MO793	Italy	KY420089		[28]
197	T. magentipunctatum	GK 4552	Greece	KY420088		[28]
198	T. magenctipunctatum	ACAMTub126	Greece	PP918865		This study
199	T. magenctipunctatum	ACAMTub381	Greece	PP918866		This study
200	T. regianum	M46	Spain	KY420100		[28]
201	T. regianum	ZB3081	Slovakia	KY420098		[74]
202	T. regianum	IC13071106	Spain	KY420103		[74]
203	T. regianum	ACAMTub226	Greece	PP918898		This study
	RUFUM
204	T. aereum	ACAMTub245	Greece	PP425915		[30]
205	T. aereum	ACAMTub467	Greece	PQ336045		This study
206	T. nitidum	AH39101	Morocco	JX402092		[75]
207	T. nitidum	MUB:Fung-0934	Spain	MN962721		[76]
208	T. rufum	SFI:TUBRUF/111208	N. Macedonia	FN433159		Unpublished
209	T. nitidum	ACAMTub039	Greece	PP918707		This study
210	T. nitidum	ACAMTub375	Greece	PP918708		This study
211	T. rufum	1798	Italy	EF362473		[77]
212	T. rufum	17114	Italy	JF908745		[78]
213	T. rufum f. ferrugineum	FHS-393	Serbia	FM205676		[14]
214	T. aff. rufum 1	ACAMTub251	Greece	PP918918		This study
215	T. rufum	1785	Italy	EF362475		[77]
216	T. rufum	FHS-XX12	Serbia	FM205690		[14]
217	Tuber sp. 57	JT13224	USA	JQ925650		[10]
218	T. aff. rufum 2	ACAMTub315	Greece	PP918919		This study
219	T. buendiae	MUB:Fung-0974	Spain	MT006095		[76]
220	T. buendiae	ACAMTub350	Greece	PP918815		This study
221	T. melosporum	AH31737	Spain	JX402095		[75]
222	T. melosporum	MA-33360	Spain	OQ866602		Unpublished
223	T. pustulatum	AQUI 9725	Spain	MK211278		[79]
224	T. rufum	MA:Fungi:28397	Spain	FM205615		[14]
225	Tuber sp.	TUF113700	Estonia	UDB027742		Unpublished
226	T. rufum f. lucidum	CMI-Unibo_4944	Iran	MW829417		[46]
227	T. rufum f. lucidum	AZ2097	Italy	FJ809888		[80]
228	T. rufum	GK4422	Greece	JX402094		[75]
229	T. aff. rufum 3	ACAMTub425	Greece	PP918727		This study
230	T. aff. rufum 3	ACAMTub067	Greece	PP918709		This study
231	T. aff. rufum 3	ACAMTub230	Greece	PP918710		This study
232	T. aff. rufum 3	ACAMTub317	Greece	PP918714		This study
233	T. zambonelliae	MUB:Fung-0995	Spain	MW632951		[81]
234	T. zambonelliae	MUB:Fung-1007	Spain	MW632955		[81]
235	T. zambonelliae	ACAMTub040	Greece	PP918921		This study
236	T. zambonelliae	ACAMTub079	Greece	PP918922		This study
	OUTGROUP
237	Choiromyces helanshanensis	HKAS 80634	China	NR153899		[82]

ITS: internal transcribed spacer; LSU: large subunit 25-28S RNA.

Table 1. Details of biological material from the genus Tuber used in the phylogenetic analyses performed in the frame of this study: the phylogenetic clade and taxon, collection code, geographic origin, GenBank accession no. for ITS and LSU sequences, and respective references. The material from which sequences were generated for the first time is presented in bold typeface, while material corresponding to type specimens is underlined.

	CLADE/Taxon	Collection Code	Origin	ITS	LSU	Reference
	AESTIVUM
1	T. aestivum	AQUI 10150	Italy	MZ423173		[32]
2	T. aestivum	S8	Slovakia	HQ285310		[39]
3	T. aestivum	2PL-M(u)	Poland	KX028767		[40]
4	T. aestivum	ACAMTub361	Greece	PP725740		[41]
5	T. aestivum	ACAMTub271	Greece	PP918676		This study
6	T. aestivum	ACAMTub376	Greece	PP918694		This study
7	T. bituminatum	AQUI 9833	Italy	OL711611		[27]
8	T. bituminatum	AQUI 8579	Greece	OL711613		[27]
9	T. bituminatum	ACAMTub310	Greece	PP918764		This study
10	T. bituminatum	ACAMTub421	Greece	PP918781		This study
11	T. magnatum	TO HG3458	Italy	MZ423175		[32]
12	T. magnatum	HHD19491	Turkey	PP239641		Unpublished
13	T. magnatum	B1	Italy	AJ586268		[42]
14	T. magnatum	ACAMTub055	Greece	PP918868		This study
15	T. magnatum	ACAMTub156	Greece	PP918870		This study
16	T. mesentericum	AQUI 9717	Italy	OL711593		[27]
17	T. mesentericum	AQUI 10238	France	OL711600		[27]
18	T. mesentericum	AQUI 10230	Greece	OL711607		[27]
19	T. mesentericum	ACAMTub111	Greece	PP918880		This study
20	T. mesentericum	ACAMTub250	Greece	PP918885		This study
21	T. panniferum	JT12835	Spain	HM485380		[43]
22	T. panniferum	MUB:Fung-0973	Spain	MN962725		[44]
23	T. panniferum	ACAMTub269	Greece	PP918896		This study
24	T. panniferum	ACAMTub385	Greece	PP918897		This study
25	T. pulchrosporum	VN091	Greece	MK113975		[29]
26	T. pulchrosporum	1961 F0388	Bulgaria	MK113982		[29]
27	T. suave	AQUI 7131	Italy	OL711623		[27]
28	T. suave	AQUI 10229	Greece	OL711627		[27]
	EXCAVATUM
29	T. excavatum	AP-T63a	Slovenia	FM205555		Unpublished
30	T. excavatum	Tub10	Italy	HM152012		Unpublished
31	T. aff. excavatum 1	ACAMTub151	Greece	PP918825		This study
32	T. aff. excavatum 1	ACAMTub162	Greece	PP918826		This study
33	T. excavatum	SFI:TUBEXC/251008B	Slovenia	FN433141		Unpublished
34	T. fulgens	AP-T60	Slovenia	FM205568		Unpublished
35	T. excavatum	zb3377	Hungary	HM152023		Unpublished
36	T. excavatum	AP-T11	Slovenia	FM205554		Unpublished
37	T. aff. excavatum 2	ACAMTub028	Greece	PP918828		This study
38	T. aff. excavatum 2	ACAMTub316	Greece	PP918829		This study
39	T. excavatum	SFI:TUBEXC/121008	Slovenia	FN433142		Unpublished
40	T. excavatum	SA1TE	Poland	KJ524533		Unpublished
41	T. aff. excavatum 3	ACAMTub168	Greece	PP918841		This study
42	T. aff. excavatum 3	ACAMTub320a	Greece	PP918851		This study
43	T. excavatum	BM100	Spain	FJ748899		[45]
44	T. excavatum	MA:Fungi:54695	Spain	FM205561		Unpublished
45	T. fulgens	zb3335_205	Hungary	HM152021		Unpublished
46	T. fulgens	CMI-Unibo_5034	Iran	MW884551		[46]
47	T. fulgens	HMT44	Italy	HM151979		Unpublished
48	T. fulgens	ACAMTub170	Greece	PP918853		This study
49	T. fulgens	ACAMTub312	Greece	PP918854		This study
50	T. iranicum	CMI-Unibo 4939	Iran	MN854634		[47]
51	T. iranicum	CMI-Unibo 4965	Iran	MN854635		[47]
	GENNADII
52	T. gennadii	B M1904	Italy	HM485361		[43]
53	T. gennadii	AH39251	Spain	JN392211		[26]
54	T. aff. gennadii	ACAMTub050	Greece	PP918857		This study
55	T. aff. gennadii	ACAMTub131	Greece	PP918858		This study
56	T. gennadii	AH38957	Spain	JN392204		[26]
57	T. gennadii	VK2141	Greece	JN392205		[26]
58	T. gennadii	AH38955	Spain	JN392207		[26]
59	T. gennadii	MRG496	Spain	OQ565278		[48]
60	T. conchae	MRG486	Spain	OQ565276		[48]
61	T. conchae	ACAMTub046	Greece	PP918816		This study
62	T. lacunosum	AH38912	Morocco	JN392214		[26]
63	T. lacunosum	MRG692	Spain	OQ565280		[48]
64	T. lucentum	j957	Spain	MN437527		[49]
65	T. lucentum	MUB:Fung-j825	Spain	NR184909		[49]
	MACROSPORUM
66	T. calosporum	HKAS:88790	China	KT444598		[50]
67	T. calosporum	HKAS:88751	China	KT444600		[50]
68	T. glabrum	BJTCFan228	China	KF002731		[51]
69	T. glabrum	BJTCFan232	China	KF002727		[51]
70	T. macrosporum	CMI-Unibo_4937	Iran	MW884553		[46]
71	T. macrosporum	CMI-Unibo_4938	Iran	MW884554		[46]
72	T. macrosporum	FHS-449	Serbia	FM205664		[14]
73	T. macrosporum	IC28092233	Spain	PP948901		Unpublished
74	T. macrosporum	ITA_014s	Italy	KP738351		[52]
75	T. macrosporum	ACAMTub052	Greece	PP918859		This study
76	T. macrosporum	ACAMTub110	Greece	PP918860		This study
77	T. macrosporum	ACAMTub252	Greece	PP918862		This study
78	T. macrosporum	ITA_003V	Italy	KP738380		[52]
79	T. macrosporum	ITA_008	Italy	KP738388		[52]
80	T. macrosporum	FRA_002	France	KP738361		[52]
81	T. monosporum	ACAMTub455	Greece	PP918907	PP892729	This study
82	T. sinomonosporum	BJTCFan150	China	KF002729		[51]
	MACULATUM
83	T. arnoldianum	RH1619	USA	KU186913		[53]
84	T. arnoldianum	FLAS:F-71902	USA	OR762689	PP337424	Unpublished
85	T. arnoldianum	FLAS:F-71880	USA	OR762672	PP337406	Unpublished
86	T. arnoldianum	AMC105	USA	OP413000		Unpublished
87	T. aztecorum	ITCV 993 ITCV	Mexico	NR159044		[54]
88	T. aztecorum	GG993 FLAS	Mexico	KY271791		[54]
89	T. beyerlei	JT32597	USA	HM485408		[43]
90	T. beyerlei	Berch 0042	Canada	KP972075		[55]
91	T. brennemanii	MES653	USA	MF611779	KY565252	[56]
92	T. brennemanii	RH1746	USA	MF611791	KY565258	[56]
93	T. excelsum-reticulatum	BJTC FAN863	China	OM265272	OM366224	[11]
94	T. excelsum-reticulatum	BJTC FAN758	China	OM265264	OM366218	[11]
95	T. foetidum	ZB2489	Hungary	JQ288906	JF261362	Unpublished
96	T. foetidum	ZB2452	Hungary	JQ288905	JF261361	Unpublished
97	T. foetidum	JCAS0140001000096	Spain	MH703905		[57]
98	T. foetidum	ZB3454	Finland	FN568055		[58]
99	T. foetidum	B-2489	Hungary	AJ557544		[13]
100	T. foetidum	B-2452	Hungary	AJ557543		[13]
101	T. leptodermum	ACAMTub475	Greece	PQ877422	PQ881954	This study
102	T. leptodermum	ACAMTub359	Greece	PP918908	PP905688	This study
103	T. leptodermum	ACAMTub379	Greece	PP918909	PP905689	This study
104	T. leptodermum	ACAMTub380	Greece	PP918910	PP905690	This study
105	T. leptodermum	ACAMTub382	Greece	PP918911	PP905691	This study
106	T. hubeiense	HMAS 60233	China	KT067688	KT067694	[11]
107	T. lusitanicum	MUB:Fung-0986	Spain	MT621651	MT705332	[59]
108	T. lusitanicum	MUB:Fung-0988	Spain	MT621653		[59]
109	T. lusitanicum	MUB:Fung-0990	Spain	MT621655		[59]
110	T. maculatum		Italy	AF003919		Unpublished
111	T. maculatum	herbarium 1967	Italy	EU753269		Unpublished
112	T. maculatum	Imatra_3	Finland	MZ389968		[60]
113	T. maculatum	FLAB22	Uruguay	PP297668		[61]
114	T. maculatum	BJTC FAN876	China	OM265278	OM366228	[11]
115	T. maculatum	BJTC FAN868	China	OM265274	OM366227	[11]
116	T. miquihuanense	ITCV885	Mexico	HM485414	JF419292	[43]; [62]
117	T. pseudomagnatum	BJTC FAN163	China	JQ771192		[63]
118	T. pseudomagnatum	BJTC FAN299	China	OM265244	OM366195	[11]
119	T. pseudomagnatum	BJTC FAN392	China	KP276184	KP276193	[64]
120	T. pseudomagnatum	BJTC FAN390	China	OM265243		[11]
121	T. pseudomagnatum	BJTC FAN389	China	OM265242		[11]
122	T. rapaeodorum	BG Kew K(M)128884	England	EU784429		[65]
123	T. rapaeodorum	CMI-UNIBO 2483	Armenia	DQ011849		Unpublished
124	T. walkeri	RH5211	USA	JF419260		[62]
125	T. walkeri	FLAS:MES-274	USA	MT156441		Unpublished
126	T. walkeri	RH794	USA	JF419258	JF419308	[62]
127	T. whetstonense	JT25783	USA	HM485392	JF419304	[43]; [62]
128	T. whetstonense	SOC 756 OSC 111412	USA	AY830855		[66]
129	T. whetstonense	FLAS:MES-210	USA	MT156439		Unpublished
130	T. wumengense	BJTC FAN218A	China	KT067682	KT067707	[64]
131	T. wumengense	HMAS 60229	China	KT067687		[64]
132	T. wumengense	BJTC FAN586	China	OM265254		[11]
	MELANOSPORUM
133	T. brumale	G1126	Greece	KF550998		[8]
134	T. brumale	BTb2p2	Turkey	KF551063		[8]
135	T. brumale	FHS-460	Serbia	FM205660		[14]
136	T. brumale	CMI-Unibo_5001	Iran	MW829419		[46]
137	T. brumale	MA:Fungi:28373	Spain	FM205550		[14]
138	T. brumale	TB59k1	France	KF550974		[8]
139	T. brumale	ACAMTub159	Greece	OP850807		This study
140	T. brumale	ACAMTub233	Greece	PP918804		This study
141	T. brumale	ACAMTub456	Greece	PP918814		This study
142	T. cryptobrumale	HNHM<HUN>:BP107922	Hungary	KU203777		[33]
143	T. cryptobrumale	HNHM<HUN>:BP107923	Hungary	KU203778		[33]
144	T. indicum	Tind-gs01	China	DQ375490		[67]
145	T. indicum	Tind-gs05	China	DQ375491		[67]
146	T. melanosporum	AQUI 10152	Italy	MZ423176		[32]
147	T. melanosporum	MEL178	France	EU555385		[68]
148	T. melanosporum	ACAMTub256	Greece	PP918878		This study
149	T. petrophilum	LJU-GIS-TUBsp_xx1010A	Serbia	HG810883		[69]
150	T. petrophilum	LJU-GIS-TUBsp_xx1010D	Serbia	HG810886		[69]
151	T. thracicum	SOMF:30882	Bulgaria	OR666020		[70]
152	T. thracicum	SOMF:30883	Bulgaria	OR666027		[70]
	PUBERULUM
153	T. oligospermum	CMI-UNIBO 4230	Morocco	KF021624		[71]
154	T. oligospermum	CMI-UNIBO 4234	Morocco	KF021622		[71]
155	T. oligospermum	AH39002	Italy	JN392248		[26]
156	T. oligospermum	AH37861	Italy	JN392251		[26]
157	T. oligospermum	GK3060	Greece	JN392249		[26]
158	T. aff. oligospermum 1	ACAMTub075	Greece	PP918887		This study
159	T. oligospermum	MUB:Fung-1049	Spain	PQ281432		Unpublished
160	T. oligospermum	MUB:Fung-1050	Israel	PQ281433		Unpublished
161	T. oligospermum	GK5388	Greece	JN392263		[26]
162	T. aff. oligospermum 2	ACAMTub062	Greece	PP918889		This study
163	T. oligospermum	HAI-D-102	Israel	JN392271		[26]
164	T. oligospermum	VK2042	Greece	JN392254		[26]
165	Soil sample	S052	Iran	UDB0765470		Unpublished
166	T. aff. oligospermum 3	ACAMTub133	Greece	PP918893		This study
167	T. anniae	JT13209	USA	HM485338		[43]
168	T. anniae	Juva_1	Finland	MZ389970		[60]
169	T. anniae	ACAMTub024	Greece	PP918746		This study
170	T. borchii	AP-T30	Slovenia	FM205497		[14]
171	T. borchii	CMI-Unibo_5047	Iran	MW829422		[46]
172	T. borchii	17Bo	Italy	DQ679802		[72]
173	T. borchii	ACAMTub135	Greece	PP918787		This study
174	T. borchii	ACAMTub334	Greece	PP918792		This study
175	T. borchii	ACAMTub371A	Greece	PP918796		This study
176	T. borchii	AQUI 10151	Italy	MZ423174		[32]
177	T. borchii	GK5597	Greece	JN392228		[26]
178	T. borchii	CMI-UNIBO 3026	Italy	FJ554493		[72]
179	T. borchii	MUB:Fung-1036	Spain	PQ273773		Unpublished
180	T. borchii	ACAMTub120A	Greece	PP918786		This study
181	T. borchii	ACAMTub207	Greece	PP918802		This study
182	T. borchii	ACAMTub377	Greece	PP918803		This study
183	T. dryophilum		Italy	AF003917		Unpublished
184	T. puberulum	GK4352	Greece	JN392223		[26]
185	T. dryophilum	GB69	Italy	HM485353		[10]
186	T. puberulum	GK5601	Greece	JN392224		[26]
187	T. dryophillum	ACAMTub043	Greece	PP918819		This study
188	T. dryophillum	ACAMTub134	Greece	PP918821		This study
189	T. dryophillum	ACAMTub182	Greece	PP918822		This study
190	T. dryophillum	ACAMTub204	Greece	PP918824		This study
191	T. lijiangense	BJTC FAN307	China	KP276188		[64]
192	T. cf. lijiangense	BJTC FAN148	China	OM286798		[11]
193	T. puberulum	Berk. & Broome TL11885	Denmark	AJ969626		[73]
194	T. puberulum	Berk. & Broome TL3857	Denmark	AJ969625		[73]
	REGIANUM
195	T. magentipunctatum	ZB4293	Hungary	JQ288909		[74]
196	T. magentipunctatum	MO793	Italy	KY420089		[28]
197	T. magentipunctatum	GK 4552	Greece	KY420088		[28]
198	T. magenctipunctatum	ACAMTub126	Greece	PP918865		This study
199	T. magenctipunctatum	ACAMTub381	Greece	PP918866		This study
200	T. regianum	M46	Spain	KY420100		[28]
201	T. regianum	ZB3081	Slovakia	KY420098		[74]
202	T. regianum	IC13071106	Spain	KY420103		[74]
203	T. regianum	ACAMTub226	Greece	PP918898		This study
	RUFUM
204	T. aereum	ACAMTub245	Greece	PP425915		[30]
205	T. aereum	ACAMTub467	Greece	PQ336045		This study
206	T. nitidum	AH39101	Morocco	JX402092		[75]
207	T. nitidum	MUB:Fung-0934	Spain	MN962721		[76]
208	T. rufum	SFI:TUBRUF/111208	N. Macedonia	FN433159		Unpublished
209	T. nitidum	ACAMTub039	Greece	PP918707		This study
210	T. nitidum	ACAMTub375	Greece	PP918708		This study
211	T. rufum	1798	Italy	EF362473		[77]
212	T. rufum	17114	Italy	JF908745		[78]
213	T. rufum f. ferrugineum	FHS-393	Serbia	FM205676		[14]
214	T. aff. rufum 1	ACAMTub251	Greece	PP918918		This study
215	T. rufum	1785	Italy	EF362475		[77]
216	T. rufum	FHS-XX12	Serbia	FM205690		[14]
217	Tuber sp. 57	JT13224	USA	JQ925650		[10]
218	T. aff. rufum 2	ACAMTub315	Greece	PP918919		This study
219	T. buendiae	MUB:Fung-0974	Spain	MT006095		[76]
220	T. buendiae	ACAMTub350	Greece	PP918815		This study
221	T. melosporum	AH31737	Spain	JX402095		[75]
222	T. melosporum	MA-33360	Spain	OQ866602		Unpublished
223	T. pustulatum	AQUI 9725	Spain	MK211278		[79]
224	T. rufum	MA:Fungi:28397	Spain	FM205615		[14]
225	Tuber sp.	TUF113700	Estonia	UDB027742		Unpublished
226	T. rufum f. lucidum	CMI-Unibo_4944	Iran	MW829417		[46]
227	T. rufum f. lucidum	AZ2097	Italy	FJ809888		[80]
228	T. rufum	GK4422	Greece	JX402094		[75]
229	T. aff. rufum 3	ACAMTub425	Greece	PP918727		This study
230	T. aff. rufum 3	ACAMTub067	Greece	PP918709		This study
231	T. aff. rufum 3	ACAMTub230	Greece	PP918710		This study
232	T. aff. rufum 3	ACAMTub317	Greece	PP918714		This study
233	T. zambonelliae	MUB:Fung-0995	Spain	MW632951		[81]
234	T. zambonelliae	MUB:Fung-1007	Spain	MW632955		[81]
235	T. zambonelliae	ACAMTub040	Greece	PP918921		This study
236	T. zambonelliae	ACAMTub079	Greece	PP918922		This study
	OUTGROUP
237	Choiromyces helanshanensis	HKAS 80634	China	NR153899		[82]

ITS: internal transcribed spacer; LSU: large subunit 25-28S RNA.

2.4. Phylogenetic Analysis

For the phylogenetic analyses, sequences generated in this study, as well as reference sequences of Tuber species derived from the GenBank (https://www.ncbi.nlm.nih.gov/genbank/; accessed on 10 March 2025) and UNITE (https://unite.ut.ee/; accessed on 10 March 2025), were used (Table 1). Sequence alignments were implemented using the ClustalW algorithm of MEGA11 software [83]. To exclude poorly aligned or ambiguous regions of the sequences, trimming was performed manually. The phylogenetic trees were constructed with the maximum likelihood (ML) method via the IQ-TREE vers. 2.4.0 software by using the GTR + gamma substitution model, estimated as the best-fit model by the same software [84,85]. ML support values in the branches were obtained using ultrafast bootstrap (1000 replicates), implemented in the IQ-TREE software [86]. Bayesian inference (BI) analysis was performed utilizing the BEAST vers. 10.5.0 software [87], employing the same nucleotide substitution model as in the ML method. The Effective Sample Sizes (ESS > 200) were evaluated by the Tracer vers. 1.7.2 software [88]. A single chain of 10 million generations was run, logging every 100 generations. A maximum clade credibility (MCC) tree was generated using TreeAnnotator vers. 10.5.0 [87] with 10% burn-in. Visualization of the phylogenetic tree was performed with the iTOL vers. 6 web-based software [89]. Bootstrap support (≥70%) and BPP (≥0.95) values are shown in the tree nodes, while Choiromyces helanshanensis (sequence from the holotype) was used as an outgroup in all cases. The tree scale indicates the expected number of changes per site. The resulting phylogenetic trees are deposited in TreeBASE (http://www.treebase.org; accessed on 20 March 2025) with accession ID 32081.

Each taxonomic annotation appearing on the phylogenetic trees corresponds to a well-defined species or to a non-described phylospecies, which is provisionally named by using the epithet of the most related taxon and the term “aff.” (affinity). Although we acknowledge the limitations mentioned by earlier studies regarding the generalized use of the ITS region as a universal DNA barcode marker for fungi, we followed the widely adopted threshold for separating species of the genus Tuber, i.e., <96% for ITS sequence identity in pairwise comparisons [10,11,27]. The respective values were estimated with the aid of the BLASTn web-based tool [90], accessed through the NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi; accessed on 10 March 2025).

3. Results

3.1. Phylogeny of the Genus Tuber

A total of 242 ITS and six LSU sequences were generated for the first time (Supplementary Material, Table S1); among them, 63 ITS and five LSU sequences were selected for inclusion in the phylogenetic trees of this study (Figure 1, Figure 2 and Figure 3, Supplementary Figure S1). In addition to the Maculatum clade, which appears compressed in the ML ITS tree (Figure 1), the following Tuber clades (as these were defined by Bonito et al. [10], and Bonito and Smith [1]) are included: Aestivum, Excavatum, Gennadii, Macrosporum, Melanosporum, Puberulum, Regianum and Rufum. They consist mostly of species of European origin and/or distribution, and contain 187 sequences (other than those of the Maculatum clade), 58 of which derive from this work and 129 of which are sequences deposited in NCBI and UNITE databases (16 of them correspond to specimens from Greece generated in the frame of previous studies) (Table 1).

The length of the aligned ITS dataset was 566 nucleotides, and the resulting phylogenetic trees inferred from ML and BI analyses demonstrated consistent topologies (with no supported conflicts) showing high ML-BS and BPP support values.

In the Aestivum clade, specimens from this study are included in five highly supported groups (ML-BS 100%, BPP 1.00) which correspond to well-defined species, i.e., T. aestivum, T. bituminatum, T. magnatum Picco, T. mesentericum and T. panniferum Tul. & C. Tul. All of them, with the exception of T. panniferum, demonstrated high sequence identity (97.2% to 100%) with the reference material examined. In contrast, T. panniferum collections from the islands of Crete and Naxos growing in association with Quercus spp. (ACAMTub243, ACAMTub269, and ACAMTub385) showed some variation (97.3–97.5% sequence identity) when compared to three sequences available in international databases.

As regards the Excavatum clade, our analysis presents six to seven phylospecies; Greek specimens are distributed in four of them, with high support (98–100%, 1.00), i.e., T. fulgens, and another three are hereby provisionally named as T. aff. excavatum 1 (ACAMTub151, ACAMTub162), T. aff. excavatum 2 (ACAMTub028, ACAMTub316) and T. aff. excavatum 3 (ACAMTub168); each of them also includes collections of European origin.

In the Gennadii clade, the existence of six phylogenetic species was detected, with high support (98-100%, 1.00). Sequences from specimens studied in this work are grouped within two of them, one being the recently described T. conchae M. Romero & P. Alvarado. The other is one of the three phylospecies consisting of specimens identified as T. gennadii; hence, our two collections from the Attica area (ACAMTub050 and ACAMTub131) are grouped within a phylospecies provisionally labelled as T. aff. gennadii, which also includes specimens from Italy and Spain.

The Macrosporum clade is divided in two main well-supported groups (100%, 1.00). One of them corresponds to the Tuber macrosporum Vittad. species complex, and is further divided into three subgroups (98–100%, 0.99–1.00); all Greek T. macrosporum specimens originate from Central and Northern Greece and grow in association with a large range of angiosperms, and are grouped into one of these subgroups. In addition, one of the collections (ACAMTub455 from Paggaio Mt. in Fagus sylvatica) studied in this work corresponds to the European T. monosporum (Mattir.) Vizzini, on the basis of morphoanatomical features alone; no reference sequence for this species exists in the international databases.

As regards the Melanosporum clade, the majority of the respective specimens from Greece are grouped within the T. brumale species complex, while others (e.g., ACAMTub256) are identified as T. melanosporum (99.6–100% sequence identity with the species epitype, MZ423176), and originate from areas in the vicinity of truffle orchards.

Greek specimens in the Puberulum clade are grouped into at least six phylospecies with high support (96–100%, 1.00): T. anniae Vittad. (98.4% sequence identity with T. anniae holotype), T. borchii Vittad. s.l. (97.5–100% sequence identity with species lectotype MZ423174), T. dryophilum Tul. & C. Tul. (98.5–99.6%, when compared to reference sequence AF003917) and the T. oligospermum complex, including three phylogenetic species provisionally named T. aff. oligospermum 1, T. aff. oligospermum 2 and T. aff. oligospermum 3. The first and second phylospecies each include one collection studied in this work (ACAMTub075—Attica area and ACAMTub062—Fthiotida area, respectively), while the third includes five collections, all from Attica (Supplementary Table S1); this material is associated with P. halepensis, P. pinea, Quercus coccifera, Q. ilex, Quercus sp. and Cistus monspeliensis.

The presence of two species of the Regianum clade was detected in Greece, i.e., T. magenctipunctatum and T. regianum. Specimens of the former demonstrated high sequence identity (99.8–100%) with the species holotype, whereas the latter is represented by a single collection (ACAMTub226, Prespes area in F. sylvatica) which showed 98.0–100% sequence identity with the six T. regianum sequences deposited in GenBank.

Regarding the Rufum clade, Greek collections were grouped into seven phylospecies. Four of them correspond to known species: T. aereum (100% sequence identity with the holotype PP425915), T. buendiae Ant. Rodr. & Morte (97.8% sequence identity with the holotype MT006095), T. nitidum and T. zambonelliae (97.6–98.1% sequence identity with the holotype, MW632951). The other three form part of phylogenetically distinct groups (100%, 1.00) within the T. rufum species complex, which are provisionally named T. aff. rufum 1, T. aff. rufum 2 and T. aff. rufum 3. The first includes one collection from this study (ACAMTub251 from Olympos Mt.), and specimens identified as T. rufum from Italy and as T. rufum f. ferrugineum from Serbia; the second consists of one Greek collection (ACAMTub315 from the Prespes area) and one other specimen, labeled as Tuber sp. 57, from the USA. The third includes 23 collections from various regions of continental Greece (Supplementary Table S1), as well as specimens from Iran and Italy, identified as T. rufum f. lucidum, and from Estonia, identified as Tuber sp.

In particular, as concerns the Maculatum clade, a total of 51 ITS and 24 LSU sequences were analyzed; among these, 5 ITS and 5 LSU sequences were generated for the first time (Table 1). For the analysis of the two loci, the final alignment included 24 specimens and spanned 1406 nucleotides, comprising 544 nucleotides from the ITS and 862 from the LSU. The tree topologies obtained from the ML and BI analysis were similar, and the former was selected for the purposes of this study.

Taxa within the Maculatum clade present a worldwide distribution, by including collections originating from Europe, Asia, and North, Central and South America. European species are represented by T. maculatum Vittad., T. foetidum Vittad., T. rapaeodorum Tul. & C. Tul. and T. lusitanicum Ant. Rodr. & Muñoz-Mohedano. Most importantly, the phylogenetic analysis of the Maculatum clade revealed the presence of a new species to science, i.e., Tuber leptodermum, which forms a monophyletic group with 100% bootstrap support and a posterior probability of 1 (100%, 1.00) in individual ITS and LSU phylogenies and in concatenated ITS+LSU phylogenies (Figure 2 and Figure 3, Supplementary Figure S1).

In the ITS and in the concatenated ITS+LSU phylogenies, T. leptodermum was resolved as a sister to T. foetidum (83%, 0.99 and 78%, 0.97, respectively), while other phylogenetically related taxa were T. hubeiense L. Fan, T. lusitanicum, T. maculatum, T. pseudomagnatum L. Fan and T. rapaeodorum. Based on a megablast search of the INSDC (GenBank) nucleotide database, the closest hits using the ITS sequence of the T. leptodermum type material were collections identified as T. foetidum from Spain (MH703905, MT621657, MT621658) and Serbia (FM205704), presenting sequence identity values of 94.6–95.7%.

3.2. Taxonomy

Tuber leptodermum V. Daskalopoulos, G. Konstantinidis, N. Tsilis, E. Polemis & G.I. Zervakis, sp. nov. (Figure 4 and Figure 5).

MycoBank: MB858411

Etymology: The epithet leptodermum derives from the Greek words “λεπτό” (“lepto”, meaning thin) and “δέρμα” (“derma”, meaning skin), and refers to the thin peridium of the new species.

Diagnosis: Ascoma medium sized, dark-brown, peridum thin, up to 150 (170) μm, pseudoparenchymatous, composed of globose-to-angular cells, exoperidium easily scratched off to reveal a light-colored endoperidium; asci 1–4-spored (rather equally varied); ascospores ellipsoid, reticulate–alveolate, with broad meshes. It is found in riparian habitats dominated by Populus and/or Salix spp. T. leptodermum is a phylogenetically distinct species in the Maculatum clade, and represents a sister taxon to T. foetidum, as revealed by the ITS and the concatenated ITS+LSU phylogeny.

Holotype: GREECE. KOZANI: Municipality of Voio, Tsotyli greater area, elevation approx. 700 m, found in soil under Populus alba, 12 October 2024, coll. N. Tsilis (holotype ACAMTub475, designated here). GenBank: ITS = PQ877422; LSU = PQ881954.

Description: Ascomata hypogeous, 20–50 mm in diameter, generally irregular, tuberiform, knotty and lobed but also subglobose, with firm and solid texture, surface felty smooth to minutely verrucose to areolate in spots, color cigar-brown {16} to dark-umber {36} or darker-brown vinaceous {25} to purplish-chestnut {21} (Figure 5a). Peridium (50) 80–150 (170) µm thick, external layer (exoperidium) (15) 25–85 (130) µm thick, pseudoparenchymatous, of subglobose to irregularly angular cells (textura globulosa-angularis), dark-brick {20} in color, without any protruding cystidioid hyphae, cells (8.5) 11–18.5 (29) × (6.5) 7.5–13.5 (16.5) µm; internal layer (endoperidium) (50) 70–110 (125) µm thick, hyaline, with relatively smaller cells in comparison with the external layer and progressively more elongated towards the gleba (Figure 5b–d). Gleba firm, solid, and whitish at first, becoming fawn {29} to umber {18} or date-brown {24} at maturity, ornamented with numerous branching white veins (Figure 5a,b). Odor intense, penetrating, reminiscent of gas mixed with a somewhat spicy aromatic scent, like horseradish (Armoracia rusticana). Taste mild and sweetish, occasionally with a mustard-like aftertaste. Asci excluding stalk (70.5) 80–109 (122.5) × (47) 58–83.5 (945) µm, Av = 94.6 × 72.3 µm (n = 100), irregularly subglobose, broadly ellipsoid or pyriform, generally without, occasionally with a short stalk measuring 33–52 × 9–18.5 µm, with 1–4 ascospores per ascus (1-spored 24%, 2-spored 37%, 3-spored 27%, 4-spored 12%, n = 500), walls approx. 1 μm thick (Figure 5e,f). Ascospores (23.0) 28.9–53.6 (61.7) × (17.8) 22.7–36.9 (42.5) µm, Av: 40.4 × 28.8, Q = (1.01) 1.23–1.57 (1.93), Qav: 1.40 (n = 400), excluding ornamentation, cinnamon-to-rusty brown at maturity, generally broadly ellipsoid-to-ellipsoid, but also sometimes subglobose, ovoid or even oblong, and reticulate–alveolate, with broad meshes, (2.4) 3.6–5.9 (10.4) µm in height; meshes regular, closed and regularly polygonal (5-6 sides), 3-5 (6) lengthwise (3 = 15%, 4 = 43%, 5 = 33%, 6 = 9%, n = 100) (Figure 5e,f). Ascospores from one-spored asci (39.8) 44.7–58.7 (61.7) × (26.1) 31.6–39.8 (42.5) µm, Av = 52 × 35.7 µm; Q = (1.2) 1.3–1.6 (1.8), Qav = 1.5 (n = 100); two-spored asci (30.6) 36.5–47.6 (53.2) × (23.5) 25.5–33.2 (40.2) µm, Av = 42.6 × 29.5 µm; Q = (1) 1.3–1.6 (1.9), Qav = 1.5 (n = 100); three-spored asci (24.5) 28.5–40.4 (47.2) × (19.4) 22.3–29.4 (33.3) µm, Av = 34.6 × 26 µm; Q = (1.1) 1.2–1.5 (1.6), Qav = 1.3 (n = 100); four-spored asci (23) 27.5–38 (43.4) × (17.8) 21.7–27.6 (29.3) µm, Av = 32.5 × 24.1 µm; Q = (1) 1.2–1.5 (1.8), Qav = 1.4 (n = 100).

Distribution and ecology: Tuber leptodermum grows in relatively humid, deciduous forests or stands of Populus, Quercus, Carpinus, Corylus and/or Salix, predominantly near water streams and lakes, in habitats where T. macrosporum and (more often) T. magnatum also appear (Figure 4). The ascomata were found from October to November, at the Kozani and Grevena prefectures in NW Greece.

Additional specimens examined: GREECE. KOZANI: under Populus, Quercus, Carpinus and Corylus, 25 November 2021, coll. P. Kladopoulou (ACAMTub359; GenBank: ITS = PP918908; LSU = PP905688); GREECE. KOZANI: under trees of the genera Populus (P. alba and P. tremula) and Quercus, 28 November 2021, coll. N. Tsilis, GK13815 (ACAMTub382; GenBank: ITS = PP918911; LSU = PP905691); GREECE. GREVENA: under Populus (P. alba, P. nigra and P. tremula), Quercus, Carpinus, Corylus and Salix, 7 November 2021, coll. I. Kalampoukas, GK13481 (ACAMTub379; ITS = PP918909; LSU = PP905689); GREECE. GREVENA: under Quercus and Salix, 17 November 2021, coll. I. Kalampoukas GK13509 (ACAMTub380; ITS = PP918910; LSU = PP905690).

Notes: T. leptodermum sp. nov. is distinct from other related species of the Maculatum clade, i.e., T. maculatum, T. rapaeodorum, T. lusitanicum, T. hubeiense, T. pseudomagnatum and T. foetidum, as demonstrated in the ITS and LSU, and in the concatenated ITS+LSU phylogenies (Figure 2 and Figure 3, Supplementary Figure S1). As regards morphology, T. leptodermum produces medium-sized (20–50 mm diam), dark-brown ascomata with a thin (up to 150 (170) μm) pseudoparenchymatous peridium, a fawn-to-umber or date-brown gleba when mature, and cinnamon-to-rusty brown ascospores with broad meshes; mature ascomata have an intense and penetrating odor reminiscent of gas mixed with a somewhat spicy aromatic scent, like horseradish. It is mostly found in riparian habitats with predominantly Populus spp. Comparisons of such features with those of the aforementioned phylogenetically related taxa reveal several differences in the size and color of ascomata, in the thickness (mainly) and/or structure of the peridium, in the presence of hair-like hyphae on the peridium surface, in the gleba color and/or in the shape, color and ornamentation of the ascospores. Especially as regards T. foetidum, which is the closest phylogenetic taxon to T. leptodermum, it is morphologically very similar to T. leptodermum, but it is distinguished from the latter by the sparse presence of very short hair-like hyphae on the peridium surface, a thicker (200–350 µm) peridium and a fetid odor [12,13].

As regards the seven Tuber species which are reported for the first time in Greece, a summary of the key morphoanatomic features of the respective collections, together with details about their distribution and ecology, are included in Supplementary Table S2. In addition, representative photos of their ascomata and microscopic characters are provided in Figure 6.

4. Discussion

The new truffle species, T. leptodermum, produces medium-sized (20–50 mm diam), dark-brown truffles which exhibit distinct morphological features, i.e. a felty smooth or sparsely warty, thin peridium (up to 150 (170) µm), pseudoparenchymatic, composed of globose-to-angular cells, and an exoperidium which is easily scratched off to reveal a light-colored endoperidium. In addition, the asci contain one to four reticulate–alveolate, ellipsoid ascospores with broad meshes. It was found to occur in riparian habitats, mainly under Populus and Salix spp., in autumn. T. leptodermum is a phylogenetically distinct species of the Maculatum clade, and is most closely related to T. foetidum, and then to T. hubeiense, T. maculatum, T. rapaeodorum, T. pseudomagnatum and T. lusitanicum, on the basis of the phylogenetic study performed.

When T. leptodermum was compared to the aforementioned species, the following differences were noted: T. hubeiense grows under Pinus armandii by forming small-sized (5–9 mm) whitish ascomata with a peridium of 180–250 µm, and yellow–brown-to-golden-brown ascospores [64]; T. lusitanicum also produces small-sized (5–20 mm) ascomata that are white at first, then pale-yellowish, sometimes with a reddish tinge, and are darker at maturity, with a thick peridium (300–500 µm) bearing sparse hair-like hyphae, an olive-brown-to-dark-brown gleba and yellowish-brown ascospores [59]; T. maculatum forms ascomata with a thick peridium (300–400 µm), prosenchymatous throughout, but often with scattered isodiametric cells or clusters of such cells, and with ellipsoid, light-golden-brown ascospores, ornamented with a regular reticulum 2–5 μm deep [91]; T. pseudomagnatum produces yellow–white ascomata with blackish gleba and small elliptic ascospores with a large meshed reticulum [63]; T. rapaeodorum produces small-sized (up to 25 mm), white-to-ochraceous or ochraceous-yellow (with dark-brown patches) ascomata with a thick peridium (250–600 µm), a snuff-brown-to-dark-purplish brown gleba and a horseradish- or radish-like odor [13].

In particular, as regards comparisons between T. foetidum and T. leptodermum, which appear to be the closest relatives (sequence identity values up to 95%), the two species demonstrate a high similarity in most of their morphoanatomical features; however, the former could be distinguished by the sparse presence of very short hair-like hyphae on the peridium surface, a thicker (200–350 µm) peridium and a fetid odor [12,13]. A phylogenetically distant species that could be confused with T. leptodermum is T. macrosporum; the latter also grows in similar (riparian) habitats, but it has is garlic-like odor and forms larger, darker colored ascospores with more meshes along the spore length.

Within the Aestivum clade, collections from our material were grouped into five species: T. aestivum, T. bituminatum, T. magnatum, T. mesentericum and T. panniferum; the presence of T. panniferum in Greece is molecularly confirmed for the first time. In addition, the occurrence of two other species of this clade, i.e., T. pulchrosporum and T. suave, has been previously recorded in various regions of the country and in the Attica area, respectively [27,29]. Especially as regards T. aestivum, a small number of seven-spored asci was noted in the majority of our collections, while a single eight-spored ascus was observed in one specimen only (Figure 7); hence, when seven collections of T. aestivum were examined, the presence of one-, two-, three-, four-, five-, six- and seven-spored asci was calculated at (percentages of total) 2.4%, 10.0%, 14.9%, 23.0%, 32.4%, 15.0% and 2.3%, respectively (n = 660). To the best of our knowledge, there is only one previous report on the presence of seven- and eight-spored asci in T. aestivum ascomata [92], despite the quite extensive pertinent literature. Noteworthy morphoanatomical variability was also detected in T. mesentericum; in measurements of ascospores in six collections, the quotient was Q = (1.01) 1.22 × 1.72 (2.17), with Qav = 1.47 (n = 613). These measurements differ substantially from the respective observations made by Leonardi et al. [27], suggesting the existence of more elongated ascospores in Greek T. mesentericum specimens. Furthermore, the latter is apparently limited to the northern part of the country, whereas T. bituminatum seems to be much more widespread and common (Supplementary Table S1).

In the phylogenetic analysis of our study, the Excavatum clade consisted of six highly supported phylospecies which included collections initially determined either as T. excavatum Vittad. or as T. fulgens Quél. (the presence of the latter is molecularly confirmed for the first time); however, none of them could be assigned with certainty to either one of these species, due to a lack of sequences from the respective type material. Most of the Greek specimens were grouped in T. aff. excavatum 3, which corresponds to “T. excavatum Clade IV” sensu Puliga et al. [47]; this phylospecies exhibits a large distribution throughout mainland Greece in coniferous, deciduous or mixed forests. Two additional phylospecies, i.e., T. aff. excavatum 1 and T. aff. excavatum 2, corresponding to “T. excavatum Clade I” and “T. excavatum Clade III” sensu Puliga et al. [47], respectively, include collections from the northern part of the country growing in association with broadleaved trees only. It should be noted that the existence of species within the Excavatum clade in Greece is molecularly confirmed for the first time.

As regards the Gennadii clade, T. conchae is reported for the first time in Greece. According to the original description (Romero and Alvarado in Crous et al. [48]), this species differs from T. gennadii because the latter “… has smaller (0.5–2 cm diam) and globose or subglobose ascomata (rarely wrinkled or lobulated), develops long cracks or locules in the gleba with age, and is probably associated to a different host plant (Tuberaria guttata)”. Interestingly, our collection’s features do not support the distinction of the two species based on these differences, since it consists of a small ascoma of ca. 2 cm diam, its gleba possesses locules and cracks, and it was found in the vicinity of T. guttata (Figure 6, Supplementary Table S2); still, the ITS sequence of the Greek collection exhibits 99.6% identity with the species holotype from Spain, leaving no doubts about its identity. It should be noted that this same T. conchae collection was previously identified as T. gennadii by using an LSU sequence only [26]. T. gennadii was described by Chatin [16] from material deriving from the Peloponnese, Greece. Recent collections from other regions of the country were examined and were identified as such together with specimens from Italy, Spain and north Morocco [23,26]; these correspond to three distinct phylospecies in our ITS tree (Figure 1). Our collections were provisionally labeled as Tuber aff. gennadii, and are grouped within one of the three aforementioned phylospecies by also including a specimen from Italy (BM1904, Genebank HM485361) which is considered to represent T. gennadii [43]. Therefore, the true phylogenetic position of this species will remain ambiguous until the holotype is sequenced.

The presence of T. macrosporum is molecularly confirmed for the first time in Greece. The respective collections are included—together with other sequences deriving from various areas of the Mediterranean Basin—in of one of the three phylogenetic groups formed with high support in the ITS tree (Figure 1); this particular group corresponds to T. macrosporum Clade I (sensu Benucci et al. [52]), while another group represents T. macrosporum Clade II, comprising sequences from France and Italy. The identity values between the sequences of T. macrosporum Clade I and II are less than 96.5%. The third group seems to be further separated, and consists of material originating from Iran [46]. Another species of the Macrosporum clade is T. monosporum, as revealed by the identity of a single collection from Greece which possesses the typical (and unique) morphoanatomical features of the species, and it was identified as such, although no pertinent sequences have been available until now in the international databases. This finding constitutes a new record for Greek mycobiota. It is worth mentioning that T. monosporum forms ascospores that differ considerably from those of other Tuber spp., since their surface has polygonal craters (like inward meshes) resembling a golf ball (Figure 6, Supplementary Table S2); therefore, it was originally placed in a distinct genus, i.e., Paradoxa monospora Mattir. However, Vizzini [93] transferred it to the genus Tuber as T. monosporum (Mattir.) Vizzini, and our phylogenetic analysis supports this placement.

As regards the Melanosporum clade in our phylogeny, it consists of six phylogroups, three corresponding to well-known species, i.e., T. brumale (Europe), T. melanosporum Vittad. (Europe) and T. indicum Cooke & Massee (China), whereas the rest represent the recently described T. cryptobrumale Merényi, T. Varga & Bratek, T. petrophilum Milenković, P. Jovan., Grebenc, Ivančević & Marković and T. thracicum Slavova, M. Leonardi, A. Paz, Assyov & Pacioni, all originating from the Balkan peninsula. Specimens from Greece are grouped either within the T. brumale species complex, or identified as T. melanosporum. Especially as regards the former, recent results indicate that the Melanosporum clade may have undergone evolutionary diversification, separating populations from eastern and southeastern Europe from those present in central and western Europe [8,9,70]. In addition, Mereneyi et al. [8] demonstrated that the “T. brumale aggregate” comprises three phylospecies corresponding to haplogroups I and II in Clade A and Clade B. The latter coincides with T. cryptobrumale [33], while haplogroup II exhibits a distribution which is limited to the wider Balkan region, including Greece, as our findings confirm; however, haplogroups I and II have yet to be formally described as distinct species. Concerning T. melanosporum, its occurrence in Greece has not been verified in areas other than those in close proximity to truffle orchards where this species is cultivated. Hence, and in the absence of other pertinent records in the country (or in neighboring regions), we believe that T. melanosporum is not a native species.

In the Puberulum clade, T. dryophilum is a relatively small, whitish and hairless truffle (or with very short hairs), with ellipsoid ascospores and a spore reticulum with very wide meshes, i.e., two to three along the spore length, as well as a plectenchymatous peridium structure [12]. However, the results of Lancellotti et al. [94] and our own observations on the respective material indicate the existence of a pseudoparenchymatic peridium with large angular cells and more rounded ascospores with 4–7 (8) meshes lengthwise (Figure 6; Supplementary Table S2). Thus, it is quite possible that specimens of T. dryophilum studied by Montecchi and Sarasini [12] correspond to another taxon from the reference collection of T. dryophilum (AF003917), as the latter is defined by Lancelotti et al. [94], and our specimens were compared with this. It is worth noting that ambiguities in discriminating the small whitish truffles of the Puberulum group s.l. (which includes the Maculatum and Gibbosum clades) are well documented, particularly by Lancellotti et al. [94], who stated that only 4 out of 44 sequences initially labeled as T. dryophilum were correctly identified.

As regards the rest of the species within the Puberulum clade, T. anniae W. Colgan & Trappe was first described in North America, growing in association with conifers. The respective Greek specimen is the first ever recorded in Greece, and it is morphoanatomically very similar to the holotype of T. anniae and other pertinent descriptions [11,95,96,97], by sharing the same pseudoparenchymatic peridium structure and the coniferous habitat (Pinus sylvestris, N. Greece). Furthermore, in our ITS phylogeny, T. borchii appears as a sister group to T. anniae (100%, 1.00; Figure 1). According to previous studies [32,72] this species consists of two “Haplotypes” with no apparent morphoanatomical or ecological differences between them. The majority of the Greek collections identified as T. borchii correspond to “Haplotype 1”, while three were grouped in “Haplotype 2” (Supplementary Table S1). As concerns T. oligospermum, the results of our phylogenetic analysis are in agreement with those of Alvarado et al. [26] by demonstrating that it corresponds to a complex of (at least) four distinct phylogenetic species with a predominantly Mediterranean distribution, three of which comprise specimens from Greece. To better distinguishing the latter, we have provisionally named them as T. aff. oligospermum 1, T. aff. oligospermum 2 and T. aff. oligospermum 3. Still, the phylogenetic grouping of T. oligospermum s.str. remains unclear, and to elucidate this issue, sequencing of the type specimen is required. Interestingly enough, our phylogenetic analysis shows that specimens previously reported as “T. puberulum” [26], i.e., GK4352 (JN392223) from Xanthi and GK5601 (JN392224) from Kozani, are grouped in T. dryophilum; thus, the presence of the latter in Greece is reported for the first time. In contrast, the existence of T. puberulum Berk. & Broome in the country has yet to be confirmed. This species was originally described from a collection found in the United Kingdom, and according to Lancellotti et al. [94], it appears almost exclusively in central and northern Europe, while it has not been recorded in Italy and in the Balkan peninsula.

The Regianum clade is composed of only three European species, i.e., T. bernardini Gori, T. megentipuncatum and T. regianum, demonstrating similar/overlapping morphoanatomic features, which makes it difficult to distinguish them by classical taxonomy only. Cseh et al. [28] reported that they could be discriminated by their relative ascospore size and volume, as well as by the average size of their ascospore meshes. However, our T. regianum collection possesses the T. magenctipunctatum ascospore pattern, both as regards size and volume. The presence of T. regianum is reported for the first time in Greece.

The Rufum clade includes some of the most taxonomically challenging groups, since it consists of many closely related (and several still undescribed) species exhibiting high phenotypic similarity, particularly within the T. rufum complex and in Mediterranean habitats [76,81,98]. In the frame of our work, the existence of T. buendiae and T. zambonelliae is recorded for the first time in Greece, thus expanding the known distribution of these species, since their presence had been reported in Spain and Morocco only. Moreover, sequences from Greek collections show a relatively high variation (up to 97.5% sequence identity values) compared to the holotypes of the two species. In addition, the existence of T. nitidum in Greece is molecularly confirmed for the first time, while T. aereum is also reported on Andros island, very close to the type locality (Naxos island; [30]). Apart from the four aforementioned taxa, our collections are grouped into three other well-supported phylospecies which were provisionally named T. aff. rufum 1, T. aff. rufum 2 and T. aff. rufum 3; the first two are recorded for the first time in Greece, while the existence of the third was also reported by Alvarado et al. [75]. Still, none of them could be assigned with certainty to T. rufum, due to absence of sequences from the respective type material. In the past, the presence of T. rufum was recorded in Greece as T. rufum var. rufum Pollini [20,21,99], as T. rufum var. apiculatum E. Fisch. [20] and as T. rufum f. lucidum (H. Bonnet) Montecchi & Lazzari [21], but it is not known yet to which of the aforementioned phylospecies these findings correspond to.

The findings of this study substantially increase our knowledge of truffle diversity, the range of associated plants, and their geographic distribution in Greece (Table 2). As a result, a total of 40 species (including 10 undescribed phylogenetic species) is now known to exist, as compared to 26 species which were previously reported, while the number of molecularly confirmed species has increased from 15 to 20.

Table 2. A summarized review of the known diversity of the genus Tuber in Greece, also including the findings of this study: taxa grouped per phylogenetic clade, associated plant(s), geographic distribution according to administrative regions of Greece, and pertinent references. Species whose presence has been confirmed by molecular approaches are presented in bold. Abbreviations for the administrative regions of Greece are as follows: East Macedonia and Thrace (EM & T), Western Macedonia (WM), Central Macedonia (CM), Epirus (Ep), Thessaly (Th), Western Greece (WG), Central Greece (CG), Attica (At), Peloponnese (P), Ionian Islands (I), North Aegean (NA), South Aegean (SA) and Crete (Cr). A map depicting the administrative regions of Greece is provided in the Supplementary Materials, Figure S2.

A/A	CLADE/Taxon	Associated Plant(s)	Distribution	References *
	AESTIVUM
1	T. aestivum Vittad. 1	Abies, Pinus, Quercus, Corylus, Cistus	EM & T, CM, WM, Ep, Th, WG, CG, At, P, SA	[18,20,21,41,100,101,102,103]; this study
2	T. bituminatum Berk. & Broome	Abies cephalonica, Abies borisii-regis, Pinus nigra, Quercus pubescens, Quercus coccifera, Quercus, Fagus sylvatica	WM, CM, Th, CG, P	[27]; this study
3	T. magnatum Picco	Populus, Carpinus, Quercus, Salix, Tilia, Corylus, Pinus	WM, CM, Th	[9,25,102]; this study
4	T. mesentericum Vittad. 1	F. sylvatica, Fagus, Corylus avelana, Corylus, Carpinus betulus, Carpinus, Quercus, Populus, Salix, Tilia, Pinus nigra, Pinus	EM & T, WΜ, CM	[27,104]; this study
5	T. panniferum Tul. & C. Tul.	Q. pubescens, Q. coccifera, Quercus ilex, Quercus	EM & T, CM, SA, Cr	[20,21,102]; this study
6	T. pulchrosporum Konstantinidis, Tsampazis, Slavova, Nakkas, Polemis, Fryssouli & Zervakis	Quercus, Corylus, Carpinus, Pinus	EM & T, WM, Ep, Th, WG, At	[29]
7	T. suave Pacioni & M. Leonardi	Q. coccifera, Pinus halepensis	At	[27]
	EXCAVATUM
8	T. excavatum Vittad.	Abies, Pinus, Quercus, Corylus, Tilia, Salix	EM & T, CM, WM, Ep, P	[20,21,99]
9	T. aff. excavatum 1	Corylus	WM	This study
10	T. aff. excavatum 2	F. sylvatica	EM & T, WM	This study
11	T. aff. excavatum 3	A. cephalonica, Q. coccifera, Q. ilex, Quercus, F. sylvatica, Carpinus, P. halepensis	EM & T, CM, WM, CG, At, P	This study
12	T. fulgens Quél.	Abies, F. sylvatica, Corylus, Carpinus, Quercus	EM & T, CM, WM, Th	[21]; this study
	GENNADII
13	T. asa Tul. & C. Tul. 2	Cistus	At	[23]
14	T. conchae M. Romero & P. Alvarado 3	Tuberaria guttata	EM & T	This study
15	T. gennadii (Chatin) Pat. 4	Helianthemum, Cistaceae	EM & T, CG, At, P	[16,21,23,26,105]
16	T. aff. gennadii	T. guttata, P. halepensis, Pinus pinea, Q. coccifera	At	This study
	MACROSPORUM
17	T. macrosporum Vittad.	Corylus avelana, Corylus, Carpinus, Quercus, Salix, Tilia, Populus	EM & T, WM, CM, Th	[24,102]; this study
18	T. monosporum(Mattir.) Vizzini	F. sylvatica	WM	This study
	MACULATUM
19	T. leptodermum Daskalopoulos, Konstantinidis, Tsilis, Polemis & Zervakis	Populus alba, Populus tremula, Populus nigra, Quercus, Salix, Corylus, Carpinus	WM	This study
	MELANOSPORUM
20	T. brumale Vittad. 5	Quercus, Carpinus, Fagus	EM & T, Ep, CM, WM, P	[8,20,21,33,104]; this study
21	T. melanosporum Vittad. 6	Quercus, Carpinus	CM	[20,21,102,104]; this study
22	T. cibarium Corda			[17]
	PUBERULUM
23	T. anniae W. Colgan & Trappe	Pinus sylvestris	CM	This study
24	T. borchii Vittad.	Abies, Pinus, Quercus, Erica	EM & T, CM, WM, Ep, Th, WG, CG, At, P, NA, SA	[20,21,26,99,102,103,104]; this study
25	T. dryophilum Tul. & C. Tul.	Quercus, Carpinus, Pinus	EM & T, WM, Th, At, P	This study
26	T. aff. oligospermum 1	P. halepensis, P. pinea, Q. coccifera	At	[26]; this study
27	T. aff. oligospermum 2	Quercus	CG	[26]; this study
28	T. aff. oligospermum 3	P. halepensis, Q. ilex, C. monspeliensis	At	[26]; this study
29	T. puberulum Berk. & Broome 7	Quercus	EM & T, WM	[20,26,104]
	REGIANUM
30	T. magentipunctatum Merényi, I. Nagy, Stielow & Bratek	A. cephalonica, C. avelana	WM, P	[28]
31	T. regianum Montecchi & Lazzari	F. sylvatica	WM	This study
	RUFUM
32	T. aereum Polemis, Daskalopoulos & Zervakis	Q. coccifera, Q. pubescens, Q. macrolepis	SA	[30]
33	T. buendiae Ant. Rodr. & Morte	A. cephalonica	CG	This study
34	T. ferrugineum Vittad.	Abies, Corylus, Quercus	CM, WM, Th, CG, At, P, NA	[21,99,106]
35	T. nitidum Vittad. 8	Quercus, Carpinus	EM & T, Ep, Th, At	[20]; this study
36	Tuber rufum Pollini 9	Abies, Quercus, Carpinus, Cistus	EM & T, WM, CM, Th, At	[20,21,99]
37	T. aff. rufum 1	Populus, Carpinus, Salix, Tilia	CM	This study
38	T. aff. rufum 2	F. sylvatica	WM	This study
39	T. aff. rufum 3  10	A. cephalonica, Pinus nigra, Pinus, Q. coccifera, Quercus, F. sylvatica	EM & T, WM, Ep, Th, CG, At, P	[75]; this study
40	T. zambonelliae Ant. Rodr. & Morte	P. halepensis, Q. ilex, Quercus, Carpinus	WM, At	This study

¹ Reported findings prior to 2021 could correspond to any one of the following species: T. aestivum, T. mesentericum, T. bituminatum and T. suave. ² The collections initially identified as T. asa [23] were later assessed as T. oligospermum [26]. ³ The collection was initially identified as Loculotuber gennadii [21], and later as T. gennadii [26]. ⁴ The collections identified as T. gennadii by Alvarado et al. [26] correspond to more than one phylospecies, which also include T. aff. gennadii. ⁵ Reported as T. brumale or T. brumale f. moschatum in the pertinent literature. ⁶ The only molecularly confirmed record of this species that does not originate directly from a truffle orchard with cultivated T. melanosporum (but in the vicinity of one) derives from this study. ⁷ The collections examined by Alvarado et al. [26] were identified as T. dryophilum by this study. ⁸ Reported as Tuber rufum var. nitidum (Vittad.) Mont. & Lazzari by Diamandis and Perlerou [20]. ⁹ Reported either as T. rufum, T. rufum var. rufum, or T. rufum f. lucidum or T. rufum var. apiculatum in the pertinent literature. ¹⁰ Reported as T. rufum by Alvarado et al. [75]. * References [8,9,26,27,28,29,30,41,75] include molecularly identified specimens.

Table 2. A summarized review of the known diversity of the genus Tuber in Greece, also including the findings of this study: taxa grouped per phylogenetic clade, associated plant(s), geographic distribution according to administrative regions of Greece, and pertinent references. Species whose presence has been confirmed by molecular approaches are presented in bold. Abbreviations for the administrative regions of Greece are as follows: East Macedonia and Thrace (EM & T), Western Macedonia (WM), Central Macedonia (CM), Epirus (Ep), Thessaly (Th), Western Greece (WG), Central Greece (CG), Attica (At), Peloponnese (P), Ionian Islands (I), North Aegean (NA), South Aegean (SA) and Crete (Cr). A map depicting the administrative regions of Greece is provided in the Supplementary Materials, Figure S2.

A/A	CLADE/Taxon	Associated Plant(s)	Distribution	References *
	AESTIVUM
1	T. aestivum Vittad. 1	Abies, Pinus, Quercus, Corylus, Cistus	EM & T, CM, WM, Ep, Th, WG, CG, At, P, SA	[18,20,21,41,100,101,102,103]; this study
2	T. bituminatum Berk. & Broome	Abies cephalonica, Abies borisii-regis, Pinus nigra, Quercus pubescens, Quercus coccifera, Quercus, Fagus sylvatica	WM, CM, Th, CG, P	[27]; this study
3	T. magnatum Picco	Populus, Carpinus, Quercus, Salix, Tilia, Corylus, Pinus	WM, CM, Th	[9,25,102]; this study
4	T. mesentericum Vittad. 1	F. sylvatica, Fagus, Corylus avelana, Corylus, Carpinus betulus, Carpinus, Quercus, Populus, Salix, Tilia, Pinus nigra, Pinus	EM & T, WΜ, CM	[27,104]; this study
5	T. panniferum Tul. & C. Tul.	Q. pubescens, Q. coccifera, Quercus ilex, Quercus	EM & T, CM, SA, Cr	[20,21,102]; this study
6	T. pulchrosporum Konstantinidis, Tsampazis, Slavova, Nakkas, Polemis, Fryssouli & Zervakis	Quercus, Corylus, Carpinus, Pinus	EM & T, WM, Ep, Th, WG, At	[29]
7	T. suave Pacioni & M. Leonardi	Q. coccifera, Pinus halepensis	At	[27]
	EXCAVATUM
8	T. excavatum Vittad.	Abies, Pinus, Quercus, Corylus, Tilia, Salix	EM & T, CM, WM, Ep, P	[20,21,99]
9	T. aff. excavatum 1	Corylus	WM	This study
10	T. aff. excavatum 2	F. sylvatica	EM & T, WM	This study
11	T. aff. excavatum 3	A. cephalonica, Q. coccifera, Q. ilex, Quercus, F. sylvatica, Carpinus, P. halepensis	EM & T, CM, WM, CG, At, P	This study
12	T. fulgens Quél.	Abies, F. sylvatica, Corylus, Carpinus, Quercus	EM & T, CM, WM, Th	[21]; this study
	GENNADII
13	T. asa Tul. & C. Tul. 2	Cistus	At	[23]
14	T. conchae M. Romero & P. Alvarado 3	Tuberaria guttata	EM & T	This study
15	T. gennadii (Chatin) Pat. 4	Helianthemum, Cistaceae	EM & T, CG, At, P	[16,21,23,26,105]
16	T. aff. gennadii	T. guttata, P. halepensis, Pinus pinea, Q. coccifera	At	This study
	MACROSPORUM
17	T. macrosporum Vittad.	Corylus avelana, Corylus, Carpinus, Quercus, Salix, Tilia, Populus	EM & T, WM, CM, Th	[24,102]; this study
18	T. monosporum(Mattir.) Vizzini	F. sylvatica	WM	This study
	MACULATUM
19	T. leptodermum Daskalopoulos, Konstantinidis, Tsilis, Polemis & Zervakis	Populus alba, Populus tremula, Populus nigra, Quercus, Salix, Corylus, Carpinus	WM	This study
	MELANOSPORUM
20	T. brumale Vittad. 5	Quercus, Carpinus, Fagus	EM & T, Ep, CM, WM, P	[8,20,21,33,104]; this study
21	T. melanosporum Vittad. 6	Quercus, Carpinus	CM	[20,21,102,104]; this study
22	T. cibarium Corda			[17]
	PUBERULUM
23	T. anniae W. Colgan & Trappe	Pinus sylvestris	CM	This study
24	T. borchii Vittad.	Abies, Pinus, Quercus, Erica	EM & T, CM, WM, Ep, Th, WG, CG, At, P, NA, SA	[20,21,26,99,102,103,104]; this study
25	T. dryophilum Tul. & C. Tul.	Quercus, Carpinus, Pinus	EM & T, WM, Th, At, P	This study
26	T. aff. oligospermum 1	P. halepensis, P. pinea, Q. coccifera	At	[26]; this study
27	T. aff. oligospermum 2	Quercus	CG	[26]; this study
28	T. aff. oligospermum 3	P. halepensis, Q. ilex, C. monspeliensis	At	[26]; this study
29	T. puberulum Berk. & Broome 7	Quercus	EM & T, WM	[20,26,104]
	REGIANUM
30	T. magentipunctatum Merényi, I. Nagy, Stielow & Bratek	A. cephalonica, C. avelana	WM, P	[28]
31	T. regianum Montecchi & Lazzari	F. sylvatica	WM	This study
	RUFUM
32	T. aereum Polemis, Daskalopoulos & Zervakis	Q. coccifera, Q. pubescens, Q. macrolepis	SA	[30]
33	T. buendiae Ant. Rodr. & Morte	A. cephalonica	CG	This study
34	T. ferrugineum Vittad.	Abies, Corylus, Quercus	CM, WM, Th, CG, At, P, NA	[21,99,106]
35	T. nitidum Vittad. 8	Quercus, Carpinus	EM & T, Ep, Th, At	[20]; this study
36	Tuber rufum Pollini 9	Abies, Quercus, Carpinus, Cistus	EM & T, WM, CM, Th, At	[20,21,99]
37	T. aff. rufum 1	Populus, Carpinus, Salix, Tilia	CM	This study
38	T. aff. rufum 2	F. sylvatica	WM	This study
39	T. aff. rufum 3  10	A. cephalonica, Pinus nigra, Pinus, Q. coccifera, Quercus, F. sylvatica	EM & T, WM, Ep, Th, CG, At, P	[75]; this study
40	T. zambonelliae Ant. Rodr. & Morte	P. halepensis, Q. ilex, Quercus, Carpinus	WM, At	This study

¹ Reported findings prior to 2021 could correspond to any one of the following species: T. aestivum, T. mesentericum, T. bituminatum and T. suave. ² The collections initially identified as T. asa [23] were later assessed as T. oligospermum [26]. ³ The collection was initially identified as Loculotuber gennadii [21], and later as T. gennadii [26]. ⁴ The collections identified as T. gennadii by Alvarado et al. [26] correspond to more than one phylospecies, which also include T. aff. gennadii. ⁵ Reported as T. brumale or T. brumale f. moschatum in the pertinent literature. ⁶ The only molecularly confirmed record of this species that does not originate directly from a truffle orchard with cultivated T. melanosporum (but in the vicinity of one) derives from this study. ⁷ The collections examined by Alvarado et al. [26] were identified as T. dryophilum by this study. ⁸ Reported as Tuber rufum var. nitidum (Vittad.) Mont. & Lazzari by Diamandis and Perlerou [20]. ⁹ Reported either as T. rufum, T. rufum var. rufum, or T. rufum f. lucidum or T. rufum var. apiculatum in the pertinent literature. ¹⁰ Reported as T. rufum by Alvarado et al. [75]. * References [8,9,26,27,28,29,30,41,75] include molecularly identified specimens.

5. Conclusions

In the frame of the study, the presence of 31 phylospecies of the genus Tuber is reported in Greece. These correspond to one new species to science (i.e., Tuber leptodermum); to 20 well-described taxa, 7 of which (T. anniae, T. buendiae, T. conchae, T. dryophilum, T. monosporum, T. regianum, and T. zambonelliae) constitute first national records; and to 10 undescribed phylogenetic species within the T. excavatum, T. gennadii, T. oligospermum and T. rufum species complexes (the existence of 6 is revealed for the first time). Collections of the T. excavatum complex are grouped into three phylospecies, while those initially identified as T. oligospermum and T. rufum appear as members of three distinct clades in each one of the respective species complexes. Moreover, sequences from Greece labeled as T. gennadii correspond to two distinct phylospecies, while the presence of T. puberulum in Greece could not be confirmed, since all specimens previously identified as such were grouped within T. dryophilum. T. melanosporum is considered non-native to the country, and T. bituminatum seems to be much more common than the phylogenetically related and morphologically similar T. mesentericum. In addition, a sequence representing T. monosporum has been generated (and becomes available) for the first time, representing a collection deriving from Northern Greece. Our results, as well as the outcome of other pertinent studies, emphasize the need for epitypification (or neotypification) of several truffle species occurring in Europe. In addition, a combined effort of truffle taxonomists could focus on the description of cryptic species within the Rufum and Excavatum clades, and among the small whitish truffles (mainly in the Puberulum and Gennadii clades), where the corresponding collections demonstrate high levels of phenotypic similarity and considerable sequence variation.