Three New Species of Tuber Discovered in Alpine Fir Forests in Yunnan, China

Three new species of Tuber, T. albicavum, T. laojunshanense, and T. umbilicicavatum belonging to the Puberulum phylogroup, are described based on specimens collected in alpine Abies forests at 3600–4000 m, Northwest Yunnan, China. T. albicavum is distinguished by its ascomata with a single chamber of 0.5–1.8 cm diameter, with an apical opening of 0.2–0.6 cm in diameter, and light golden-brown alveolate reticulate ascospores up to 30 μm in length; T. laojunshanense is characterized by having ascomata with a slightly tomentose surface, sometimes with a white navel, a relatively thick peridium, up to 280 µm, and yellow-brown spores with alveolate reticulate ornamentation, up to 34 µm in length; T. umbilicicavatum is characterized by smooth ascomata with a distinct white navel, a relatively thin peridium, up to 110 µm, and golden or golden-brown alveolate reticulate ascospores, up to 40 μm in length. The molecular analysis of the internal transcribed spacer region also supports that these three new species differ from previously described Tuber species.

Notably, species such as T. melanosporum Vittad., 1831, and T. magnatum Picco, 1788, are among the most expensive delicacies in the world.Since the 1990s, species like T. sinense K. Tao & B. Liu, 1989, T. sinoaestivum J.P. Zhang & P.G.Liu, 2013, and T. pseudohimalayense G. Moreno, Manjón, J. Díez & García-Mont., produced in China, have become commercially significant truffles.In southwest China, the hunting and trading of these truffles have become vital sources of income.Recently, new species of Tuber have been discovered in southwest China, with some entering the trading market, attracting considerable attention.
Since the description of the first Chinese truffle species, T. taiyuanense B. Liu, in 1985, more than sixty truffle species have been reported in China [4,[19][20][21], with expectations of more discoveries.This paper describes three new Tuber species recently found under alpine fir forests in Northwest Yunnan, China.

Materials and Methods
The specimens were collected from the alpine Abies forrestii var.smithii Viguié & Gaussen forests in Northwest Yunnan, China.These specimens were included with other studied specimens and were deposited at the BMDLU (Biological Science Museum of Dali University) and KUN-HKAS (Herbarium of Cryptogams Kunming Institute of Botany, Academia Sinica), China.

Morphological Study
Descriptions of microscopic and macroscopic characters were based on specimens (BMDLU L20065, L20066, L21218a, HKAS131251, 131252, 131253, 131254, 131255, 131256, 131257, 131258), following the methods of Kumar et al. [22] and Truong et al. [23], and mycorrhizal specimens (HKAS131253-ECM) following the methods of Agerer [24] and Janowski & Leski [25].Macroscopic characters of ascomata and gleba were observed under a Nikon SMZ1000 stereo zoom microscope.The sections were made with a razorblade by hand, mounted in a 5% KOH solution or water.The sections were observed under a light microscope.The temporarily prepared microscope slides were placed under magnification up to 1000× using Nikon ECLIPSE80i (Nikon, Tokyo, Japan) compound stereomicroscope for observation and microscopic morphological photography.Measurements were made using the Image Frame work v.0.9.7.To represent variation in the size of basidiospores, 5% of measurements were excluded from each end of the range and extreme values were given in parentheses [4].In the taxonomic descriptions of species, 'Q (L/I)' refers to the length/width ratio of ascospores in side-view; 'Q m ' refers to the average Q of all ascospores ± standard deviation; 'n' refers to the number of spores measured.Key colors were obtained from Kornerup and Wanscher [26].

DNA Extraction, PCR Amplification, and Sequencing
Total genomic DNA was extracted from the specimen using the OMEGA Plant Genomic DNA Kit.The internal transcribed spacer (ITS) rDNA region was amplified with PCR primers ITS1F and ITS4 [23,27,28].The large subunit nuclear ribosomal DNA (LSU) region was amplified with the PCR primers LROR and LR5 [29].Each 30 µL PCR mixture contained 15 µL 2 × Taq Plus Master Mix II (Sangon Biotechnology Co., Kunming, China), 13 µL ddH 2 O, 0.5 µL 10 µM of forward and reverse primers, 1 µL DNA.PCR reactions were performed on a BIO-RAD C1000TM instrument.Thermal cycles with the following settings: initial denaturation for 5 min at 94 • C, followed by 32 cycles of 40 s denaturation at 94 • C, annealing at 56 • C for 40 s for ITS, and 52 • C for 30 s for LSU, extension for 1 min at 72 • C, and final extension at 72 • C for 10 min.The PCR products were verified on 1% agarose electrophoresis gels stained with ethidium bromide.The purification and sequencing of the PCR products was conducted by Sangon Biotech Limited Company (Shanghai, China).

Sequence Alignment and Analysis
ITS was used for the analysis of Tuber species diversity in this study because they appear as a useful locus for the delimitation of the genus.Ninety-nine ITS sequences from NCBI and this study representing 54 species of Tuber (Table 1), including Labyrinthomyces sp., Choiromyces alveolatus, and Choiromyces meandriformis as outgroups (Figure 1).All ITS sequences were extracted from ascomata of Tuber specimens except one extracted from ECM.Sequences of Tuber species generated in this study were submitted to the GenBank database.We first edited the sequences using BioEdit v. 7 [30], then used the basic local alignment search tool for the GenBank database to recheck whether the newly generated sequences were amplified DNA from contaminant or not and examined clusters with closely related sequences.DNA sequences were retrieved and assembled using SeqMan.Sequences were aligned using MAFFT version 7 [31].Maximum likelihood (ML) analysis was performed using RAxML-HPC2 v. 8.2.12 [32] as implemented on the Cipres portal [33], with the GTR + G + I model and 1000 rapid bootstrap (BS) replicates for all genes.A reciprocal 70% bootstrap support approach was used to check for conflicts between the tree topologies from individual genes.As the topology of the ML tree and the Bayesian tree are similar, the ITS1, ITS2, and 5.8S sequences were combined using SequenceMatrix [34], partitioned phylogenetic analyses.For Bayesian inference (BI), the best substitution model for each partition was determined by MrModeltest 2.2 [35].The results suggested that ITS1: JC + I, 5.8S: GTR + G + I, ITS2: K80 + I + G. Bayesian analysis was performed using MrBayes ver.3.2.7a[36] on the Cipres [33]; four parallel runs were performed for 10 million generations sampling every 100th generation for the single gene trees.Parameter convergence > 200 was verified in Tracer v. 1.7 [37].The phylogenetic clade was strongly supported if the bootstrap support value (BS) was ≥70% and/or a posterior probability (PP) < 0.01.

Phylogenetic Analysis
The ML and Bayesian analyses of the 99 ITS sequences are shown in Figure 1 with associated bootstrap supports for branches.
In the phylogenetic tree, the 99 ITS sequences from Tuber ascomata revealed the phylogenetic relationship of 54 species: Clade 1 includes seven sequences of new species T. laojunshanense ascomata and one sequence of ectomycorrhizae formed by T. laojunshanense and Abies forrestii var.smithii from China.Clade 2 includes two sequences of new species T. umbilicicavatum from China.Clade 3 includes three sequences of new species T. albicavum from China.They belong to the Puberulum phylogroup.We selected the sequences of similar species of the genus Tuber distributed in China, and the sequences of species belonging to the Puberulum phylogenetic group for phylogenetic analysis with our collected specimens.The phylogenetic analysis showed that the new species are distinct from other Tuber species.
In addition to the ITS sequences used in this phylogenetic analysis, the LSU sequences were amplified from the newly supplemented specimens in this study and uploaded to NCBI for future study.
Etymology: albicavum, refers to the ascomata having a white interior chamber.
Peridium 80-140 µm thick, composed of two layers: outer layer pseudoparenchymatous, 27.0-62.5µm thick, composed of subglobose to subangular cells of 6.5-14.0(-18.0)µm wide, hyaline, thin-walled; the cells in the outermost layer expand into bristle-like outer hyphae, 0.5-1.0µm diam.at the broadest part of the base, needle-like heads, some are perpendicular to the surface, some are intertwined and prostrate, and occasionally with yellow-brown (5B4) pigment; the inner layer consists of hyaline interwoven hyphae, 29.6-74.4µm thick, the boundary between the inner and outer layers gradually transitions, with the cells of the outer layer becoming smaller.The interior of the chamber is composed of hyaline interwoven hyphae, 50.2-80.6µm thick, many hyphae extend beyond the surface, giving it a white, fluffy appearance, outer hyphae stubby, blunt head, with septa, occasionally forked, 45.2-76.2µm long, 1.0-1.5 µm diam.
Ecology and distribution: Hypogeous, solitary, or in groups in the soils under the forest of Abies forrestii var.smithii, alt.3700-3800 m, fruiting from autumn.Known only from Yunnan Province, China.
Edibility: fragrant, edible.Notes: The phylogenetic tree shows that Tuber albicavum is closely related to the known species T. tomentellum, T.liui, and a new species reported in this study, T. umbilicicavatum, forming the same clade.Compared to them, firstly, in terms of macroscopic characteristics, T. albicavum has a basal depression that forms a cavity, while T. umbilicicavatum only presents a navel-like depression.The ascomata of T. tomentellum are merely described as having 'an indistinctly basal depression' [21,58], and ascomata of T. liui have grooves and very small pores, with white soft hairs within the grooves [58].Secondly, the ascomata surface of T. albicavum is even and finely tomentose, which is similar to T. tomentellum, but T.liui and T. umbilicicavatum have smooth ascocarp surfaces.Additionally, both T. tomentellum and T. albicavum are found in Yunnan Province, China, but T. tomentellum is distributed in Pinus forests at altitudes not exceeding 2000 m [21], while T. albicavum is found in Abies forrestii var.smithii forests at altitudes of 3800-3900 m.T. liui, which also occurs in high-altitude regions (3100 m), is found in the alpine Quercus aquifolioides Rehder & E. H. Wilson forests [58].Molecular analysis also shows that T. albicavum is separated from other Tuber species, they were divided into different species with a high support rate.
Edibility: fragrant, edible.Notes: The phylogenetic tree shows that Tuber.laojunshanense is closely related to T. liyuanum and a potential Tuber species found in Taiwan, forming the same clade.When comparing the two, T. liyuanum [47] and T. laojunshanense have similar colored ascomata, which are light brown or light khaki, with a similar surface characterized by shallow irregular fissures and slight tomentose cover.They also have a similar peridium thickness and a two-layered structure.However, the obvious differences are that T. liyuanum has larger ascospores reaching 60 µm in length, while T. laojunshanense has smaller ascospores, only up to 34 µm in length.Furthermore, T. liyuanum has a strong or pungent but pleasant scent when fresh, whereas T. laojunshanense has a light pleasant scent when fresh.Additionally, both T. liyuanum and T. laojunshanense are found in Yunnan Province, China, but T. liyuanum is distributed in Pinus yunnanensis Franch.forests at altitudes not exceeding 2000 m [47], while T. laojunshanense is found in Abies forrestii var.smithii forests at altitudes of 3600-3800 m.Molecular analysis also shows that T. laojunshanense is separated from other Tuber species; they were divided into different species with a high support rate.

MYCOBANK MB 851759
Diagnosis: Differs from other Tuber spp.by its smooth ascomata with a distinct white navel, a relatively thin peridium up to 110 µm, and golden or golden-brown alveolate reticulate ascospores up to 40 µm length.
Peridium 80-110 µm thick, composed of two layers: outer layer pseudoparenchymatous, 29.5-47.5 µm thick, composed of subglobose to subangular cells of 8.5-30 µm wide, hyaline, thin-walled, occasionally cells of the outermost layer with light brown (4B6) pigment; inner layer of hyaline interwoven hyphae, 27.7-52.2µm thick, the boundary between the inner and outer layers is gradually transitioned by the cells of the outer layer becoming smaller.

Discussion
Since the first Tuber species was documented in China in 1985, more than sixty species have been reported, with half of these being newly identified by science [4,20,21].The great majority of these species are found in southwest China (Yunnan, Sichuan, and Ecology and distribution: Hypogeous, solitary, or in groups in the soils under the forest of Abies forrestii var.smithii, alt.3800-3900 m fruiting in autumn.Known only from Yunnan Province, China. Additional specimen examined: China, Yunnan Province, Lijing, Jiuhe Town, 26 • 29 ′ N, 99 • 39 ′ E, alt.3916 m, 19 September 2021, Lin Li (GenBank: HKAS131257 ITS = PP151576 LSU = PP151586).
Edibility: fragrant, edible.Notes: The phylogenetic tree shows that Tuber umbilicicavatum is closely related to T. liui and T. tomentellum, forming the same clade.Comparing the three, all have small ascomata not exceeding 3 cm, but the distinguishing feature of T. umbilicicavatum is its smooth surface with a distinct navel-like depression.In contrast, T. liui also has a smooth surface but with grooves and very small pores, and the grooves contain white soft hairs [58], T. tomentellum has a tomentose surface with an indistinct navel [21].Additionally, the ascospores of T. umbilicicavatum are smaller, up to 40 µm in length, whereas T. tomentellum ascospores can be 70 µm in length [16], and T. liui ascospores can reach 78 (-94) µm in length [58].Furthermore, T. tomentellum is distributed in Pinus forests at an altitude of around 2000 m in central Yunnan [21], T. liui is found in Quercus aquifolioides forests at an altitude of 3100 m, and T. umbilicicavatum is distributed in Abies forrestii var.smithii forests at altitudes of 3800-3900 m.Molecular analysis also shows that T. umbilicicavatum is separated from other Tuber species; they were divided into different species with a high support rate.

Discussion
Since the first Tuber species was documented in China in 1985, more than sixty species have been reported, with half of these being newly identified by science [4,20,21].The great majority of these species are found in southwest China (Yunnan, Sichuan, and Xizang Province) implying southwest China might be one of the epicenters for the evolution of Tuber species.Quite a few alpine Tuber species have been found in southwest China, which further supports this speculation.T. liui was the first alpine species found in the Quercus aquifolioides forest at alt. 3100 m, Xizang of China [58].T. zhongdianense was the second one discovered at 3400 m in Quercus monimotricha bush in Yunnan, China [59].T. albicavum, T. laojunshanense, and T. umbilicicavatum were recently found under the alpine fir forests in northwest Yunnan, growing with Abies forrestii var.smithii at even higher altitudes between 3600 and 4000 m.All five species belong to the Puberulum phylogroup sharing the same morphological features: smaller light-colored ascomata, double-layer peridium, and alveolate reticulum ascospores.Phylogenetic analysis showed all the alpine species were grouped, indicating they are closely related in phylogeny.The unique climate of the alpine zone forests in southwest China nurtured these truffles and made them differ from other Tuber species.
In a molecular-based study on the symbiotic tree partners of Tuber species [7], 16 European Tuber species were analyzed, including 156 ECM symbionts formed by Tuber species.None of the Tuber species were found to be exclusively associated with trees of the Pinaceae family, reflecting the diversity of the mycorrhizal tree partners among the Tuber species.The study also revealed that, for species within the Puberulum phylogroup (specifically T. borchii and T. anniae), approximately 30% of ECM symbionts were formed with coniferous trees (Pinaceae), while about 70% were formed with broad-leaved trees [7].
The three new species reported in this paper also belong to the Puberulum phylogroup, and the habitats of the studied specimens are similar.Although these species were confirmed to be associated with Abies forrestii var.smithii, it is known that the same Tuber species can form symbiotic mycorrhizae with different trees in various habitats, including both coniferous and broad-leaved trees.Therefore, it cannot be conclusively stated that these species form mycorrhizal associations exclusively with Abies forrestii var.smithii.Nevertheless, these truffles play an important role in the alpine forest ecosystems, symbiotically associated with their trees, and provide food to animals dwelling in these forests.

Conclusions
Based on morphological and DNA sequence evidence, this study describes three new species of white truffles, T. albicavum, T. laojunshanense, and T. umbilicicavatum, col-

Figure 1 .
Figure 1.Phylogeny derived from a maximum likelihood (ML) analysis of the nrDNA-ITS sequences from Tuber species, using Choiromyces alveolatus, C. meandriformis, and Labyrinthomyces sp. as outgroup.Values next to nodes reflect maximum likelihood bootstrap support values (BS), left, and Bayesian posterior probabilities (PP), right.Names of novel species and samples with newly generated sequences are in bold.

Table 1 .
Taxa information and GenBank accession numbers of the sequences used in this study.The newly generated sequences are in bold.