First Record of Summer Truffle (Tuber aestivum) in Portugal

Abstract

Tuber aestivum, commonly known as the summer truffle, is typically found in various parts of Europe where it grows naturally. However, its presence in Portugal was not confirmed until now. The first fruit bodies were collected in April 2024 at stone pine stands (Alenquer and Arruda dos Vinhos, Lisbon) and in June at holm oak stands (Salir, Faro). These specimens are characterized by hypogeous, subglobose, black ascomata with a peridium surface covered with pyramidal warts. Ascopores are subglobose-to-broadly ellipsoid, distinctively ornamented, usually 1–6 per asci. According to the results of the internal transcribed spacer (ITS) rDNA sequence analysis, these specimens form a well-supported group within the Aestivum clade, with T. aestivum being the closest phylogenetic taxon. This remarkable discovery opens up new opportunities for truffle exploitation in Portugal thanks to the summer truffle’s gastronomical value and high market prices.

1. Introduction

Recently, numerous new species of Tuber have been identified and recognized as valid species by the scientific community. It is difficult to assure how many Tuber species occur worldwide, since the Global Biodiversity Information Facility (GBIF) estimate that there are around 230 accepted species [1], but the Catalogue of Life only list 196 accepted species [2]. To estimate the number of Tuber species on the European continent, various sources were consulted and compared, based on material samples or preserved specimens’ records [1,2,3,4]. These sources suggest that 51 Tuber species have been found in Europe, namely T. aestivum (Wulfen) Spreng., T. albidum Fr., T. alcaracense Ant. Rodr. and Morte, T. anniae W. Colgan and Trappe, T. asa-foetida Lesp., T. bellonae Quél., T. borchii Vittad., T. brumale Vittad., T. cistophilum P. Alvarado, G. Moreno, Manjón, Gelpi and Jaime Muñoz, T. cryptobrumale Merényi, T. Varga and Bratek, T. davidlopezii Ant. Rodr., Morte and Muñ.-Moh., T. dryophilum Tul. and C. Tul., T. excavatum Vittad., T. ferrugineum Vittad., T. foetidum Vittad., T. fulgens Quél., T. gennadii (Chatin) Pat., T. gibbosum Harkn., T. himalayense B.C. Zhang and Minter, T. huidongense Y. Wang, T. lacunosum Mattir., T. levissimum Gilkey, T. lucentum Bordallo, T. lusitanicum Ant. Rodr. and Muñoz-Mohedano, T. macrosporum Vittad., T. maculatum Vittad., T. magentipunctatum Merényi, I. Nagy, Stielow and Bratek, T. magnatum Picco, T. malacodermum E. Fisch., T. malenconii Donadini, Riousset, G. Riousset and G. Chev., T. melanosporum Vittad., T. mesentericum Vittad., T. microsporum Vittad., T. mutabile Quél., T. nitidum Vittad., T. oligospermum (Tul. and C. Tul.) Trappe, T. panniferum Tul. and C. Tul., T. petrophilum Milenković, P. Jovan., Grebenc, Ivančević and Marković, T. pseudobrumale Y. Wang and Shu H. Li, T. pseudoexcavatum Y. Wang, G. Moreno, Riousset, Manjón and G. Riousset, T. puberulum Berk. and Broome, T. pulchrosporum Konstantinidis, Tsampazis, Slavova, Nakkas, Polemis, Fryssouli and Zervakis, T. rapaeodorum Tul. and C. Tul., T. regianum Montecchi and Lazzari, T. rufum Pollini, T. scleroneuron Berk. and Broome, T. scruposum R. Hesse, T. sphaerospermum (Malençon) P. Roux, Guy García and M.C. Roux, T. stramineum Ferry and Quél., T. suave Pacioni and M. Leonardi, T. vesicoperidium L. Fan.

According to GBIF, only eight Tuber species occurred in Portugal: T. borchii, T. excavatum, T. lacunosum, T. maculatum, T. oligospermum and T. puberulum, with less than 40 records in total. Although, T. gennadii [5], one of the most common truffles in Portugal, is not referred. In short, from all databases, there is no reference of the occurrence of T. aestivum in Portugal.

Tuber aestivum, commonly known as the summer truffle or Burgundy truffle, is highly prized worldwide for its distinctive aroma and flavor. Summer truffles have a milder, more delicate aroma compared to the more intense winter truffles, and its scent is often described as earthy, nutty, and slightly garlicky [6]. High demanded in gourmet and fine dining sectors, summer truffle can be used fresh or in infusions (oils, butter, cheeses) in a wide variety of prepared dishes. The economic value of summer truffle is influenced by its demand versus its availability, as well as by the ascocarps quality, size, and freshness. Fresh summer truffle market prices can vary widely between regions, with lower values in Spain (25 to 70 EUR per kg) and higher in Switzerland (200 to 600 EUR per kg) [7]. Additionally, summer truffles have medicinal properties, such as anti-angiogenic and anti-inflammatory activities [8]. In summary, T. aestivum holds significant gastronomical and economic value due to its unique flavor, culinary versatility, and medicinal properties. Although, not as costly as some other truffle varieties, it remains a luxury item that commands a premium price on the market.

T. aestivum belong to Tuberaceae (Pezizales, Ascomycota), a large group of ectomycorrhizal fungi growing in symbiosis with the roots of several vascular plant (Angiosperms: Betula spp., Carpinus spp., Castanea spp., Corylus avellana, Fagus spp., Populus spp., Quercus spp. and Tilia spp.; Gymnosperms: Abies spp., Cedrus spp., Picea spp. and Pinus spp.), fruiting abundantly at matures stands [9,10,11,12]. The T. aestivum fruit body is hypogeous, dark, globose with an average diameter of 7 cm and not easy to find without trained animals. The optimal soil conditions for its growth and development include specific ranges for soil pH (7.0 to 8.0), organic matter content (2 to 4%), and a C:N ratio (20:1 to 30:1) [12], mainly from loam to silt loam sandy–clayey soils [13]. Concerning climatic conditions, T. aestivum prefers mild temperatures (mean annual temperature between +7 °C and +11 °C (max. +18 °C) and moderate precipitation (annual precipitation between 700 mm and 900 mm (min. 400 mm)) [13,14].

T. aestivum natural occurrence exhibits a broad range distribution through Europe, Asia, and North Africa (Figure 1); however, its cultivation is wider and includes other continents. In Portugal, several edible mushrooms and truffles are collected for both personal consumption and retail sale. Mushroom and truffle hunters are typically rural individuals with a deep, intuitive knowledge gained from years of experience. They rely entirely on their expertise, without the aid of animals, like pigs or dogs, to assist in their mushroom search. It is important to note that many hypogeous sporocarps cannot be identified solely by observing the visual characteristics of the terrain or the presence of their host species, as they are buried more than 10 cm deep, leaving no visible trace of their presence. In such cases, the assistance of trained animals to detect them by their scents is necessary. Bearing this in mind, several mycological expeditions with trained dogs were performed in the last years, and many hypogeous fungi were collected (Santos-Silva, unpublished data), some of them novelties for Portugal. Although the authors had identified various Tuber species, both fructifications and mycelium, none of them were from the T. aestivum group. The main goal of the present study was to reveal, for the first time, the occurrence of the summer truffle in Portugal, and hypothesize about the reasons behind the late detection of this species in this territory. This finding, apart from its ecological importance, opens new opportunities to explore this delicacy in Portuguese gastronomy and can also become an economic asset for the country.

2. Materials and Methods

2.1. Truffle Collection and Location

Truffles were collected between April and June 2024, with the assistance of trained dogs, at 3 different locations, in the center (Alenquer and Arruda dos Vinhos) and southern (Salir, Faro) Portugal. Putative tree hosts and organoleptic features of the collected truffles were registered. Fresh truffle specimens were photographed in the field and brought to the laboratory for morphological studies and molecular analysis. Small fragments of each specimen were frozen at −20 °C for DNA amplification, and the remaining were dried at 40 °C and stored in sealed plastic bags, labeled with collection details. The samples were deposited at the Évora University Herbarium (UEVH-FUNGI), Portugal.

2.2. Morphological Study

The external characteristics of ascocarp, including shape, color, and appearance, were recorded in detail from fresh specimens. The ascomata were then dissected, and the morphology of the peridium and gleba was described. Microscopic examination was performed using distilled water and KOH 5%. Spore dimensions were determined based on the measurements of randomly selected (≈100) mature spores, outside asci, mounted in distilled water. Asci and ascospores were examined using a Leica DM750 microscope equipped with a digital camera (Leica ICC50W, Wetzlar, Germany). For species-level determination, ascomata features were compared with descriptions provided by Leonardi et al. [10].

2.3. DNA Extraction, ITS Amplification and Sequencing

Genomic DNA was extracted from approximately 100 mg of fresh ascocarp tissue using the Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research, Freiburg, Germany), following the manufacture’s protocol. The ribosomal internal transcribed spacer (ITS) region was amplified via a polymerase chain reaction (PCR) using standard primer pairs ITS4 and ITS5 primers [25]. Each PCR reaction was performed in a total volume of 25 μL, containing 1× DreamTaq^TM Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA), 7.5 pmol of each forward (ITS5) and reverse primers (ITS4), and approximately 20 ng of total DNA. The PCR program for ITS region amplification was as follows: initial denaturation at 95 °C for 3 min, followed by 30 cycles of 95 °C for 30 s, 54 °C for 45 s, and 72 °C for 1 min, with a final extension at 72 °C for 5 min. PCR products were visualized on 1% agarose gels in 1× TAE buffer, stained with GreenSafe Premium (NZYtech Lda, Lisbon, Portugal), and the GeneRuler 100 bp Plus DNA ladder (Thermo Fisher Scientific, Waltham, MA, USA) was used as a molecular weight marker. The PCR products were purified using the DNA Clean & Concentrator kit (Zymo Research, Freiburg, Germany), and sequenced using the Sanger method with the ITS4 primer by Stab Vida (Lisbon, Portugal).

2.4. Phylogenetic Analyses

The sequence reads were analyzed and edited using BioEdit v.7.2.5 [26]. Trimmed sequences were deposited in the GenBank of the National Center for Biotechnology Information (NCBI) under accession numbers PP928456 to PP928462. For phylogenetic analysis, reference ITS sequences representing the closest relatives of the Tuber aestivum were selected, and the sequences of high nucleotide similarity were retrieved from the GenBank using the BLAST algorithm [27]. A total of 70 Tuber ITS rDNA sequences were used for phylogenetic analysis by including six new sequences of T. aestivum and 64 sequences from GenBank, which correspond to 22 Tuber taxa mainly belonging to Aestivum clade and of European distribution (

Table 1. Details of the ITS sequences used for the construction of the phylogenetic tree.

Species	Sample Code	GenBank Accession No.	Origin
Tuber aestivum	S1/2TA	KJ524524	Poland
Tuber aestivum	BG9TA	KJ524523	Poland
Tuber aestivum	TA-GS	KC330220	Poland
Tuber uncinatum	01UNC	FM205618	Spain
Tuber aestivum	I1TA	KJ524528	Italy
Tuber aestivum	HKAS70296	JQ348412	France
Tuber aestivum	E61	AF516783	Italy
Tuber aestivum	HKAS70297	JQ348413	France
Tuber aestivum	E2	AF516781	Italy
Tuber aestivum	E5	AF516791	Italy
Tuber aestivum	E24	AF516792	Italy
Tuber aestivum	HKAS70292	JQ348411	Sweden
Tuber aestivum	HKAS70291	JQ348410	Sweden
Tuber aestivum	2SL	KX028765	Slovakia
Tuber uncinatum	228	AJ492199	Italy
Tuber aestivum	1SL	KX028764	Slovakia
Tuber aestivum	NW1TA	KJ524527	Poland
Tuber aestivum	E17	AY226042	Italy
Tuber aestivum	E60	AF516789	Italy
Tuber aestivum	UEVH 2005855	PP928456	Portugal
Tuber aestivum	UEVH 2005856	PP928457	Portugal
Tuber aestivum	UEVH 2005859	PP928458	Portugal
Tuber aestivum	UEVH 2005862	PP928460	Portugal
Tuber aestivum	UEVH 2005863	PP928461	Portugal
Tuber aestivum	UEVH 2005864	PP928462	Portugal
Tuber sinoaestivum	HKAS59100	JN896356	China
Tuber sinoaestivum	HKAS59101	JN896357	China
Tuber sinoaestivum	HKAS59102	JN896358	China
Tuber sinoaestivum	HKAS59111	JN896352	China
Tuber mesentericum	ZB 2228-BPU	OL711606	Hungary
Tuber mesentericum	AQUI 6558	OL711588	Greece
Tuber mesentericum	TmsW088-W047	AJ888044	Sweden
Tuber mesentericum	UASWS1873	KY197989
Tuber mesentericum	HKAS70294	JQ348414	France
Tuber mesentericum	M1	AF516794
Tuber mesentericum	M13	AF516793
Tuber pulchrosporum	GK3801	MK113979	Greece
Tuber pulchrosporum	VK4482	MK113980	Greece
Tuber pulchrosporum	1945 F8517	MK113981	Bulgaria
Tuber pulchrosporum	1961 F0388	MK113982	Bulgaria
Tuber panniferum	JT12835	HM485380	Spain
Tuber malenconii	02MLC	FM205597	Spain
Tuber malenconii	17110	JF908743	Italy
Tuber magnatum	JT19460	HM485374	Italy
Tuber magnatum	Ma2	AF106888	Italy
Tuber magnatum	GB12	JQ925645	Italy
Tuber magnatum	B6	AJ586271	Italy
Tuber magnatum	GO2	AJ586251	Croatia
Tuber macrosporum	HUN_001H	KP738366	Hungary
Tuber macrosporum	ITA_011A	KP738395	Italy
Tuber pustulatum	AQUI 9725	MK211278	Spain
Tuber pustulatum	AQUI 9728	MK211282	France
Tuber malacodermum	JT32319	FJ809889	Spain
Tuber melosporum	AH31737	JN392144	Spain
Tuber umbilicatum	T2_HKAS44316	GU979031
Tuber huidongense	SDBR-CMU-MTUF006	KT758730	Thailand
Tuber rufum	06RUF	FM205602	Slovenia
Tuber rufum	TUBRUF/141207	FM205636	Slovenia
Tuber ferrugineum	MUB:Fung-0972	MN962719	Spain
Tuber brumale	T.bru-eu03	DQ329360	France
Tuber brumale	GB52	HM485345	Italy
Tuber brumale	GB53	FJ748900	Italy
Tuber indicum	GB236	FJ748906	China
Tuber melanosporum	A3	AF106873	Italy
Tuber pseudoexcavatum	HKAS 39504	AY514310	China
Tuber spinoreticulatum	RH158	GQ221454	USA
Tuber excavatum	M1TE	KJ524534	Poland
Tuber excavatum	NW1TE	KJ524535	Poland
Tuber gennadii	B M1904	HM485361	Italy
Tuber gennadii	A JT9627	HM485360	Spain
Outgroup
Choiromyces venosus	AH38915	JF300146	Italy
Choiromyces venosus	AH38904	JF300147	Romania
Choiromyces magnusii	AH19770	JF300143	Spain

Sequences newly generated in this study are in bold.

). Choiromyces venosus (Fr.) Th. Fr. (JF300146, JF300147) and C. magnusii (Mattir.) Paol. (AH19770) were used as the outgroup.

Sequence alignment was performed using the online version of the multiple-sequence alignment program MUSCLE 3.8.425 [28] and checked manually. The alignment comprised 1249 characters, with 550 parsimony-informative sites. This generated alignment was used for phylogenetic analyses and evolutionary model selection using IQ-TREE v 1.6.12 [29,30]. The best evolutionary model for the ITS marker, as selected by ModelFinder [31], was TNe + I + G4. Both maximum likelihood (ML) and Bayesian methods were used for phylogenetic analyses. The resulting tree is the consensus of 1000 replicates of two phylogenetic analyses; an ultra-fast maximum likelihood analysis [32] with 1000 bootstrap replicates, complemented with a Bayesian approximation branch support analysis [33]. The branch lengths of consensus tree were optimized by maximum likelihood on original alignment. The tree was visualized and edited using iTOL v 6.9.1 [34]. The Robinson–Foulds distance between the ML tree and the consensus tree of 2 indicates that no significant conflicts were found between the ML and Bayesian consensus tree topologies.

3. Results

3.1. Taxonomical Characterization

Morphological and anatomical features of the collected specimens agreed with those provided by Leonardi et al. [10] (Figure 2).

Family Tuberaceae Dumort.

Genus Tuber P. Micheli ex F.H. Wigg.

Tuber aestivum (Wulfen) Spreng.—Portugal, Arruda dos Vinhos (Lisboa), under Pinus pinea L. stand, 30-IV-2024, sample n. 1015, UEVH 2005855, GenBank PP928456; Portugal, Arruda dos Vinhos (Lisboa), under Pinus pinea L. stand, 20-V-2024, sample n. 1016, UEVH 2005856, GenBank PP928457 (Figure 2A); Portugal, Alenquer (Lisboa), under Pinus pinea L. stand, 26-V-2024, sample n. 1019, UEVH 2005859, GenBank PP928458; Portugal, Alenquer (Lisboa), under Pinus pinea L. stand, 26-V-2024, sample n. 1020, UEVH 2005860, GenBank PP928459; Portugal, Salir (Loulé, Faro), under Quercus rotundifolia Lam. stand, 09-VI-2024, sample n. 1022, UEVH 2005862, GenBank PP928460 (Figure 2B); Portugal, Salir (Loulé, Faro), under Quercus rotundifolia Lam. stand, 09-VI-2024, sample n. 1023, UEVH 2005863, GenBank PP928461; Portugal, Salir (Loulé, Faro), under Quercus rotundifolia Lam. stand, 09-VI-2024, sample n. 1024, UEVH 2005864, GenBank PP928462.

3.2. Phylogenetic Analyses

Phylogenetic analyses show that our specimens are assigned to the T. aestivum nest within the Aestivum clade, which is congruent with the morphological analyses (Figure 3). The consensus tree of Bayesian approximation and maximum likelihood resolves the T. aestivum/T. uncinatum as an independent, monophyletic and well-supported clade. This species appears as the sister clade of T. sinoaestivum. In addition, the ITS sequence of two Portuguese specimens, T. aestivum voucher UEVH 2005863 and T. aestivum voucher UEVH 2005864, had a 100% nucleotide similarity with the ITS sequence of the T. aestivum strain E17 from Italy, while the ITS sequence of T. aestivum voucher UEVH 2005859 and T. aestivum voucher UEVH 2005862 had a 100% nucleotide similarity with the T. aestivum strain E60 from Italy.

4. Discussion

The Aestivum clade comprises species associated with a wide range of host plants and is found in Europe, North Africa, and Asia [35], and most recently in America [1]. Notable species include T. aestivum Vittad. (the type species of this clade), T. panniferum Tul. and Tul., T. malenconii Donadini, Riousset, G. Riousset and G. Chev., and T. mesentericum Vittad., T. sinoaestivum Zhang and Liu from China, T. magnatum Picco and along with the recently described T. pulchrosporum Konstantinidis, Tsampazis, Slavova, Nakkas, Polemis, Fryssouli and Zervakis. Tuber aestivum (Wulfen) Spreng. (including T. uncinatum Chatin) and T. sinoaestivum J.P. Zhang and P.G. Liu specimens can be easily distinguished macroscopically by their blackish peridial surface with prominent pyramidal warts and ascospores bearing a complete reticulum. Both macro- and microscopical characteristics align with the T. aestivum descriptions provided by Leonardi et al. [10]. Additionally, molecular analysis revealed that the specimens collected in Portugal cluster with the T. aestivum specimens from other locations, confirming their identification as T. aestivum. Therefore, surprisingly, this is the first report of T. aestivum in mainland Portugal based on morphological and molecular analyses.

Several factors might explain why this is the first time the summer truffle has been reported in Portugal. One possible explanation is the use of trained dogs, which detect truffles underground without any surface indication. However, it is important to note that the initial signs of truffle presence were observed due to soil disturbance caused by wild boars, which unearthed some truffles. Another possible explanation is the exceptional weather conditions preceding the current fruiting season. Precipitation from September 2023 to May 2024 was above the climatological norm for six out of nine months (Figure 4). Notably, October 2024 was the fourth wettest month since 1941. Additionally, air temperatures during this period were higher than average. It is thus likely that the unusually warm and wet weather conditions likely contributed to the atypical fruiting of T. aestivum in 2024, which, in combination with animal behavior (wild boars), allowed for the detection of these species. Another possible explanation for the detection of summer truffle only in the present year could be related to shifts in habitat conditions or climate change, which might have made these regions suitable for the presence of this fungus and consequently its fruit body development.

More studies are being conducted in areas where T. aestivum has been discovered to determine the biotic and abiotic soil conditions that may explain the appearance, persistence, and fruiting of summer truffles. These studies could also provide insights into other potential locations for these truffles. Targeted field trips with trained dogs will follow to investigate the presence of T. aestivum and identify its possible hosts. Pursuing this line of research can reveal new ecological relationships between truffles, their host trees, and other organisms, and deepen our understanding of the intricate web of life.