China • Yunnan Province: Xiangyun County (25°20'768"N, 100°43'34"E), in soil under mixed forest, with Pinus yunnanensis dominant, 20 Sep 2020, S. H. Li L3694 (holotype YAAS L3694, paratype YAAS L3695).

ON454668 (ITS); ON428904 (LSU); OQ305202 (RPB2); OQ305199 (TEF1); ex paratype: OQ297680 (ITS).

“Marroninum” refers to the maroon color of the ascoma.

Tuber marroninum differs from other species by its whitish and glabrous ascomata and maroon color of the ascoma, globose and subglobose or broad ellipsoid ascospores, with greyish-white alveolate-reticulate ornamentation.

Ascomata (Fig. 3a, b) 3–5 cm diam, subglobose, hypogeous, whitish (3A1-2) becoming brown (8E6-8) when bruised, surface smooth, with white furrows; odor mild, taste not recorded. Peridium (Fig. 3c, d) two-layered, 150–200 µm thick, outer layer 40–60 µm thick, pseudoparenchymatous, composed of subglobose or irregularly shaped cells of 7.5–16.8 (24.5) µm broad with thickened walls, inner layer composed of thin-walled hyaline interwoven hyphae, 2.5–4 (5.5) µm diam, large cells of 20 × 10 µm diam. Sometimes, intermixed. Gleba (Fig. 3b) solid, firm, brown to dark brown (10F6-8) at maturity, marbled with whitish (1A1-1B1) narrow veins. Asci (Fig. 3e, g) subglobose or irregular, 1–4 spored, with a short stalk, 50–75 (100) × 40–55 (80) μm (n = 50). Ascospores (Fig. 3e, g) subglobose to globose, pale yellow (5A2-3, 5B3) when young, becoming brown (5C6-8, 5D6-7) at maturity, excluding their spiny-reticulate ornamentation, 41.5–52 × 41–50 μm, Q = 1.00–1.14 (n = 55), in one-spored asci, 33–41 × 32–40 μm (n = 50), Q = 1.00–1.15, in two-spored asci, 21–37 × 20–35.5 μm, Q = 1.01–1.12 (n = 60), in three-spored asci, 20–35.5 × 18.5–30.5 μm, Q = 1.02–1.15 (n = 60), in four-spored asci, Q = 1.14 ± 0.07, with a greyish white (2B1-2; 1C1-2) reticulatum and alveolate-reticulate ornamentation, meshes 5–8 across the ascospore width, 1.5–3 μm tall.

Figure 3. 

Photographs of Tuber marroninum (YAAS L3694, holotype): a fresh ascomata showing cut section b the gleba c, d vertical section of peridium d the gleba and asci e, f ascospores g scanning electron micrograph of ascospore.

China: Yunnan province, Xiangyun county, hypogeous, in the soil in mixed woods dominated by Pinus yunnanensis.

Tuber marroninum is phylogenetically closely related to T. caoi (Fan et al. 2016). In morphology, T. caoi has gray and pubescent ascomata, but T. marroninum has whitish and glabrous ascomata. Both trees of ML and BI (Figs 1, 2) also have strong bootstrap supporting the new species.

China • Yunnan Province: Changning County (38.3903°N, 102.3535°E), in soil under mixed forest, with Pinus yunnanensis dominant, 20 Sep 2020, S. H. Li L3385 (holotype YAAS L3385, paratype YAAS L3682).

ON454665 (ITS); ON428901 (LSU); ex paratype: ON454667 (ITS).

Conditum, Latin, refers to the acrid smell of ascocarp.

Tuber conditum differs from other species by having no cystidia on surface of ascomata, thinner peridium, and larger ellipsoid ascospores.

Ascomata (Fig. 4a) 3–4 cm diam., subglobose, hypogeous, grey-white to grey-brown (4A2-4; 4B3-4), surface smooth, with a few pinholes; acrid smell, taste not recorded. Peridium (Fig. 4b) one-layered, 120–200 µm thick, prosenchymatous, composed of interwoven hyphae 1.5–2.5 µm broad with thin-walled, and lumens 1.5–2.5 (3.5) µm diam, with ellipsoid or irregular cells of 15 × 10 μm diam. Sometimes intermixed, wall thickness 1–2 μm. Gleba (Fig. 4a, c) solid, firm, brown to black (6E7-8; (5-9)F8) at maturity, marbled with whitish narrow veins. Asci (Fig. 4d–e) subglobose or irregular, 1–4 spored, hyaline, thin-walled or occasionally with walls as thick as 2 μm, sessile stalk, 55–100 × 45–75 μm (n = 30). Ascospores (Fig. 4d–f) subglobose or ellipsoid, pale yellow (2A2-4), yellow-brown (5D6-8; 5E7-8) at maturity, excluding ornamentation, 49–66.5 × 34.5–60 μm, Q = 1.02–1.58 (n = 54), in one-spored asci, 33–54.5 × 30–47 μm, Q = 1.06–1.35 (n = 50), in two-spored asci, 21.5–44 × 21–37 μm, Q = 1.00–1.26 (n = 60), in three-spored asci, 20.5–40.0 × 20–35.5 μm, Q = 1.01–1.30 (n = 40), in four-spored asci, Q = 1.15 ± 0.23, reticulate ornamentation 2–5 μm in height, composed of irregular hexagonal meshes, 6–8 along the spore length and 3–6 along the breadth.

Figure 4. 

Photographs of Tuber conditum (YAAS L3385, holotype): a fresh ascomata showing cut section b vertical section of peridium c, d the gleba and asci e ascospores f scanning electron micrograph of ascospore.

China: Yunnan province, Changning county, hypogeous, in the soil in woods dominated by Pinus yunnanensis.

Tuber conditum is phylogenetically closely related to Tuber tomentosum (Kinoshita et al. 2011), T. qujingense, and T. songlu (Wan et al. 2021). However, T. conditum has no cystidia on the surface of ascomata and larger ellipsoid ascospores, differing from T. tomentosum (Kinoshita et al. 2011; Sasaki et al. 2016). Tuber conditum differs from T. qujingense and T. songlu by its thinner peridium (120–200 µm) and larger ascospores (49–66.5 × 34.5–60 μm in one-spored asci). Tuber qujingense has a greyish-white ascomata, brown snowflake-shaped gleba, and prosenchymatous peridium, fusiform, and T. songlu has dense spine-like dermatocystidia, which are completely different from each other. In addition, DNA sequence analysis revealed the similarity between two species is less than 82.73% (T. qujingense) and 93% (T. songlu) in ITS sequences, strongly supporting the separation of T. conditum from the Chinese species of T. qujingense and T. songlu. Phylogenetic analysis (Figs 1, 2) also suggested that T. conditum is a distinct species because all sequences of this new species clustered within a strong bootstrap (BS ≥ 99% and PP = 1.0). The truffle has the acrid smell, which might be a good identifying feature for the fungus.

The description of Tuber pseudoexcavatum Y. Wang et al. (1998) was originally based on two specimens collected in China and published by Wang et al. in 1998. Subsequently, additional collections (AH 18387, AH 18331) from China were successfully used to generate DNA data, which were deposited in the Herbarium of the Institute of Forestry and Soil Sciences. This study reports two new collections that suggest the presence of this species may not be uncommon in the southwestern region of China. Morphologically, T. pseudoexcavatum is characterized by subglobose ascocarps with a deeply excavate appearance, ranging in color from brown to brown-orange. The surface exhibits a coarsely warted texture, while the asci contain 1–8 ascospores with spinoreticulate ornamentation, which may serve as diagnostic features based on the current collections. The known distribution of this species is limited to China.

Location • Yunnan Province, Changning County (37.1212°N, 101.7307°E). Habitat: Hypogeous, found in soil beneath a pure Pinus armandii forest Altitude: Approximately 2500 m Collection Date: 20 Sep. 2020 Specimens: YAAS L3932, YAAS L3933 Collector: S. H. Li Repository: Herbarium of Institute of Forestry and Soil Sciences (YAAS).

Tuber calosporum S. P. Wan et al. was originally described based on four specimens collected from Huize County in Yunnan Province, as detailed by Wan et al. in 2016. The specimens (wsp352, wsp145, wsp186, and wsp382) provided successful DNA data. This study introduces two additional collections, suggesting a broader distribution of the species in the southwest region of China. Morphologically, T. calosporum is recognized by its brownish-yellow ascocarps with a verrucose surface, featuring superficial yellow furrows. The ascospores are characterized by their large, ellipsoid shape and shallow alveolar ornamentation, which can serve as diagnostic traits based on the present collections. The species is known to occur exclusively in China.

Location • Yunnan Province, Xiangyun County (25°39'43"N, 100°52'27"E) Habitat: Found in soil beneath a mixed forest dominated by Pinus yunnanensis Collection Date: 20 Sep 2020 Specimens: YAAS L3648, YAAS L3684 Collector: S. H. Li Repository: Herbarium of Institute of Forestry and Soil Sciences (YAAS).