Tuber torulosum: A new truffle species decorated with moniliform cystidia from Japan

Abstract

We describe a new truffle species, Tuber torulosum, based on molecular and morphological analyses. This species forms a single globose ascospore per ascus, pale yellow in color, as do Japanese T. flavidosporum and Chinese T. turmericum and T. xanthomonosporum in the Japonicum clade of the Tuber phylogeny. However, it can be distinguished from them microscopically by its whitish tomentose mycelium that partially covers the ascoma surface and the mesh size of its spore ornamentation. Cystidia are moniliform and yellowish to reddish. Molecular phylogenetic analysis using the internal transcribed spacer and partial large subunit regions of ribosomal DNA also supports T. torulosum as a distinct species. On the basis of our results, we provide a key to species in the Japonicum clade.

The genus Tuber is an ectomycorrhizal ascomycetous taxon that belongs to the Pezizales. All members of this genus form symbiotic associations with forest tree species in families such as Fagaceae and Pinaceae and produce hypogeous fruiting bodies that are known as truffles (Hall, Brown, & Zambonelli, 2007). Some species of truffles are sought after as gourmet foods (e.g., the black truffle T. melanosporum Vittad. and the white truffle T. magnatum Pico), and the discovery of new taxa is expected to contribute to our understanding of fungal diversity and may provide new food resources.

In our previous phylogenetic study of Japanese Tuber specimens, we found that the genus contains 20 phylotypes, two of which form a novel lineage: the Japonicum clade (Kinoshita, Sasaki, & Nara, 2011). The clade is composed of two Japanese species, T. japonicum H. Sasaki, A. Kinosh. & Nara and T. flavidosporum H. Sasaki, A. Kinosh. & Nara (Kinoshita, Sasaki, & Nara, 2016), and two Chinese species, T. turmericum L. Fan (Fan, Liu, & Cao, 2015) and T. xanthomonosporum Qing & Y. Wang (Qing et al., 2015). A defining spore character of this group is that asci contain one or two globose ascospores that are pale yellow in color.

During our mycological survey in 2015, we collected samples of truffle ascomata that are pale yellow to brown, the gleba of which is mostly occupied by single-spored light yellow asci; they appeared to be similar to members of the Japonicum clade. We here describe the morphological characters of these specimens and the results of molecular analyses.

Three ascomata samples were collected from the A soil horizon by raking the humus layer under Castanea crenata Siebold et Zucc. trees in a Fagaceae-dominated forest located in Miyagi Prefecture, Japan. Morphological observation was performed for fresh and dried ascomata specimens. For macroscopic characteristics, we observed ascoma size, peridial structure, and color following the Munsell system, mostly using fresh specimens. Microscopic features of all three fresh and dried specimens were observed in water and in 3% (w/v) KOH solution. Photographs were taken under a differential interference contrast microscope (AXIO Imager A1; Carl Zeiss, Göttingen, Germany), and the size of mature ascospores and asci, peridium thickness, and other microscopic features were determined using PhotoRuler 1.1 (http://hyogo.inocybe.info/_userdata/ruler/help-eng.html). For scanning electron microscopy, spores were scraped from the dried gleba, pasted directly onto a specimen stub, and photographed with a scanning electron microscope (Miniscope TM-1000; Hitachi, Ltd., Tokyo, Japan). The specimens were deposited in the Mycological Herbarium of Forestry and Forest Products Research Institute Herbarium (TFM) and the National Museum of Nature and Science (TNS) in Japan.

We performed DNA extraction, PCR amplification, and DNA sequencing according to the procedures described in Kinoshita et al. (2018). Total DNA was extracted from small pieces (1 mm³) of glebal tissue of fresh or dried specimens using a DNeasy Plant Mini Kit (Qiagen, Valencia, California). We sequenced the nuclear ribosomal internal transcribed spacer (ITS) and the partial 28S nuclear rDNA (28S) including the D1 and D2 regions for phylogenetic analyses. To amplify the ITS region, the primer pairs ITS1F (Gardes & Bruns, 1993) and ITS4 (White, Bruns, Lee, & Taylor, 1990) were used. To amplify the partial 28S region, the primer pairs LR0R and LR5 (Vilgalys & Hester, 1990) were used. DNA amplification was performed by using the TaKaRa Multiplex PCR kit ver.2 (Takara Bio Inc., Shiga, Japan). PCR products were purified with ExoSAP-IT reagent (Affymetrix, Santa Clara, California) and bidirectionally sequenced using the same primers that were used for PCR amplification. Sequencing was performed using an ABI3500 automated sequencer (Applied Biosystems, Foster City, California) with a BigDye Terminator 3.1 Cycle Sequencing Kit (Applied Biosystems) following the manufacturer's instructions.

BLAST searches (blastn: https://blast.ncbi.nlm.nih.gov; Altschul et al., 1997) were conducted for the ITS and partial LSU of nuclear rDNA sequences against the International Nucleotide Sequence Database (INSD, DDBJ: http://www.ddbj.nig.ac.jp/; EMBL: http://www.ebi.ac.uk/; GenBank: http://www.ncbi.nlm.nih.gov/), respectively. The closest match sequences with ≥ 80% sequence identity for ITS and ≥ 90% for LSU sequences were retrieved from the GenBank database (Table 1). The ITS and LSU datasets were aligned using MAFFT 7 (Katoh & Standley, 2013) with the default settings. Poorly aligned sites were identified and cleaned by using the software Gblocks 0.91b (Castresana, 2000) with all parameters left at default values. The resultant ITS and LSU matrixes were used for maximum likelihood (ML) analyses with RAxML-NG (Kozlov, Darriba, Flouri, Morel, & Stamatakis, 2019) under a TIM2+I+G4 nucleotide substitution model for ITS and a GTR+I+G4 nucleotide substitution model for LSU selected by ModelTest-NG (Darriba et al., 2019). One thousand ML bootstrap replicates were computed in RAxML-NG to evaluate branching support. The ML tree was visualized with iTOL (Letunic & Bork, 2006). Bayesian phylogenetic analyses were also conducted using MrBayes 3.2.7 software (Ronquist et al., 2012). After setting the best substitution models as determined by ModelTest-NG (GTR+G+I model for both ITS and LSU), two independent MCMC chains were run, sampling every 1000th tree until the standard deviation of the split frequency (ASDSF) became < 0.01. The log files of MrBayes were further analyzed using Tracer 1.7 (Rambaut, Drummond, Xie, Baele, & Suchard, 2018) to check the effective sample sizes, which were always > 200, indicating sufficient independent sampling to estimate the posterior distribution of each parameter. The first 10% of the sampled trees were discarded as burn-in. The remaining trees (1622 for ITS and 2306 for LSU) were used to construct a 50% majority rule consensus tree, and the consensus trees were visualized with iTOL (Letunic & Bork, 2006). The ITS and LSU alignment files were deposited in TreeBASE (http://treebase.org/treebase-web/; Accession No. S26573).

Table 1. List of ITS and LSU sequences retrieved from the GenBank databases and used in the phylogenetic analysis

Taxon	Origin	Voucher	GenBank
			ITS	LSU
Choiromyces alveolatus	USA	HS2886	HM485333
Choiromyces alveolatus	USA	MES97	HM485332
Choiromyces helanshanensis	China	80645		KP019361
Choiromyces meandriformis	USA	GB285	HM485331
Geomorium fuegianum	Chille	CT-4392		MK430984
Reddelomyces westralinensis	Australia	OSC:111640		GQ231748
Tuber borchii	Italy	GB32		FJ809799
T. borchii	Italy	GB45	HM485344
T. borchii	Italy	GB62	HM485342
T. californicum	USA	JT22590	HM485351
T. californicum	USA	JT8207	HM485352
T. caoi	China	BJTC FAN309		KP276199
T. cf. anniane	USA	JT22986		FJ809851
T. excavatum	Unknown	Trappe 19457		DQ191677
T. ferrugineum	Hungary	ZB3363		MT270600
T. flavidosporum	Japan	TFM: S16012 (holotype)	AB553446	AB553520
T. fulgens	Italy	M2435		JQ925691
T. gibbosum	USA	AFTOL-ID 1344		FJ176877
T. gibbosum	USA	JT26632	FJ809862
T. gibbosum	USA	JT6555	FJ809863
T. huizeanum	China	BJTC FAN186		KT067703
T. huizeanum	China	BJTC FAN186 (holotype)		NG_059991
T. japonicum	Japan	H1	AB553432
T. japonicum	Japan	K460	AB553442
T. japonicum	Japan	TFM: S16001 (holotype)	AB553444	AB553519
T. japonicum	Japan	TFM: S16003 (paratype)	AB553433
T. japonicum	Japan	TFM: S16004 (paratype)	AB553434
T. japonicum	Japan	TFM: S16005 (paratype)	AB553437
T. japonicum	Japan	TFM: S16006 (paratype)	AB553438
T. japonicum	Japan	TFM: S16007 (paratype)	AB553440
T. japonicum	Japan	TFM: S16008 (paratype)	AB553441
T. japonicum	Japan	TFM: S16009 (paratype)	AB553439
T. japonicum	Japan	TFM: S16011 (paratype)	AB553445
T. jinshajiangense	China	BJTC FAN407		KX575847
T. latisporum	China	HKAS 44315 (holotype)	DQ898183
T. lijiangense	China	BJTC FAN307		KP276203
T. liyuanum	China	BJTC FAN162		KT067698
T. lyonii	USA	GB 112		EU394704
T. maculatum	Denmark	TK5974	AJ969627
T. maculatum	Italy	A15	AM406673
T. microspiculatum	USA	OSC 62169		NG_042662
T. multimaculatum	Spain	OSC:62169		NG_042662
T. nitidum	Hungary	ZB3914		MT270604
T. oregonense	USA	JT15112	FJ809881
T. oregonense	USA	JT30493	JQ925647
T. pacificum	USA	OSC 62159		KT968651
T. parvomurphium	China	BJTC FAN298 (holotype)		NG_059981
T. pseudomagnatum	China	BJTC FAN299		KP276193
T. pseudomagnatum	China	FAN163	JQ771192
T. pseudosphaerosporum	China	BJTCFan250	KF744063
T. rufum	Spain	TR70		MT270602
T. shearii	USA	OSC51052	HM485389
T. sinosphaerosporum	China	BJTC:FAN135	JX092086
T. sinosphaerosporum	China	BJTC:FAN136	JX092087
T. sphaerosporum	USA	JT12487		FJ809853
T. subglobosum	China	BJTC:FAN153		MH115322
T. thailandicum	Thailand	CMU-MTUF1		KP196333
T. thailandicum	Thailand	CMU-MTUF2		KP196334
T. torulosum	Japan	TNS-F-91471 (holotype); TFM: S-15001 (isotype)	LC637314	LC637317
T. torulosum	Japan	TNS-F-91472; TFM: S-15002 (paratype)	LC637315
T. torulosum	Japan	TNS-F-91473; TFM: S-15003 (paratype)	LC637316
T. turmericum	China	BJTC FAN459	KT758834
T. turmericum	China	BJTC FAN471	KT758835
T. turmericum	China	BJTC FAN472 (paratype)	KT758836
T. turmericum	China	BJTC FAN473 (holotype)	KT758837
T. turmericum	China	BJTC FAN474	KT758838
T. turmericum	China	BJTC FAN475	KT758839
T. turmericum	China	BJTC FAN476	KT758840
T. turmericum	China	BJTC FAN477	KT758841
T. turmericum	China	BJTC FAN482	KT758842
T. umbilicatum	China	BJTC:FAN225		HM115326
T. vesicoperidium	China	L155		JQ690068
T. xanthomonosporum	China	YAAS L3185 (holotype)	KJ162154
T. xanthomonosporum	China	YAAS L3186	KJ162155
T. xanthomonosporum	China	YAAS L3187	KJ162156
T. zhongdianense	China	BJTC FAN178		KT067701
Uncultured ectomycorrhiza	China	XS99	KX444500

Taxonomy

Tuber torulosum A. Kinosh., Koh. Yamam. & A. Yamada, sp. nov. Fig. 1.

MycoBank no.: MB 840190.

Fig. 1 - Tuber torulosum (TFM: S15001, holotype). A: Ascomata. B: Mycelial mats (h) and soil particles (s) entangled by hyphae on the ascoma surface. C: Vertical section of peridium (p, peridium; g, gleba). D: Bead-like moniliform cystidia arising from the peridium. E: Scanning electron micrograph of the cystidia. F: Light micrograph of ascospores. The paired micrographs show the same field at a different focal plane, i.e. upper surface (left) and middle plane (right). G: Scanning electron micrograph of an ascospore.

Diagnosis: Tuber torulosum forms monosporic asci with moniliform cystidia on the peridium. The species differs from the closely related T. flavidosporum, T. turmericum, and T. xanthomonosporum by having fewer and wider meshes on the ascospore surface.

Type: JAPAN, MIYAGI Prefecture, Kawasaki-machi, under Castanea crenata trees, 15 Dec 2015, collected by Kohei Yamamoto (holotype, TNS-F-91471; isotype, TFM: S-15001).

DNA sequences: holotype LC637314 (ITS), LC637317 (LSU)

Etymology: torulosum (Lat.), referring to the moniliform cystidia on the ascomata surface

Japanese name: Juzudama-seiyoshoro from Juzudama = beads, which refers to the cystidia that form on the ascomata, seiyoshoro = Japanese name for the genus Tuber).

Ascomata subglobose to irregular shaped, 6‒16 mm diam, yellowish white (10YR 7/4), odor indistinct in both fresh and dried specimens. Peridium yellowish white with reddish tomentum in patches, engaging the soil particles, cystidia with a moniliform shape, thick-walled, yellow or olive, 200-250 µm thick, composed of two layers: the outer layer 100-150 µm thick and pseudoparenchymatous, composed of irregularly shaped cells 15‒25 × 8‒17 µm, pale yellow or hyaline; the inner layer 70‒100 µm thick, of complex interwoven cells. Gleba yellow to olive, marbled with white sterile veins, few voids. Asci spherical, mostly 1-spored, very rarely 2-spored, 96‒111 × 57‒70 µm (n = 50), obovate to broadly ellipsoid. Ascospores 32‒45 µm (n = 50) in diam. excluding reticulate ornaments of 4‒8 µm in height, composed of mostly irregular hexagonal meshes 15‒17 × 10‒15 µm, 3 across the spore width, globose, light yellow.

Habitat and distribution: Known only from the type locality under a transplanted C. crenata tree from within Japan in Quercus-dominated forest. Soil type is Andosols and ocher-yellow clay at the A and B soil layers, respectively.

Specimens examined (paratypes): JAPAN, MIYAGI Prefecture, Kawasaki-machi, under C. crenata trees, 15 Dec 2015, leg. Kohei Yamamoto (TNS-F-91472; TFM:S-15002); Ibid., 15 Dec 2015, leg. Kohei Yamamoto (TNS-F-91473; TFM:S-15003).

The ITS sequence matrix contained 47 sequences and 730 aligned bases, of which 281 bp were identified as poorly aligned by Gblocks. All poorly aligned sites were excluded before phylogenetic analyses. The resultant ITS alignment was 449 bp long, with 242 constant, 207 variable, and 190 parsimony-informative sites. The LSU dataset included 34 sequences and 943 aligned bases, of which 55 bp were identified as poorly aligned by Gblocks. The resultant LSU alignment was 888 bp, with 234 variable and 159 parsimony-informative sites. An examination of log-likelihood in Tracer 1.7 indicated that the MCMC chains reached convergence. The tree inferred from the ML analyses is shown in Fig. 2.

Fig. 2 - A RAxML tree of Tuber torulosum and related taxa based on rDNA ITS sequences. ML bootstrap values and Bayesian posterior probabilities are indicated on branches as MLBS/BPP. Values supported by both BS ≥ 75% and PP ≥ 0.95 are shown.

Both ML and Bayesian analyses of ITS sequences showed four well-supported lineages within the Japonicum clade (shown as ML bootstrap values / Bayesian posterior probabilities in Fig. 2). The three sequences of T. torulosum form a clade with one environmental sequence from an oak (Quercus liaotungensis) ectomycorrhizal root sampled in China (KX444500), with strong support (100/1.00). Sister to this clade, with strong branch support (94/1.00), is a group that contains a single sequence of T. flavidosporum and a clade composed of T. turmericum and T. xanthomonosporum sequences. Nucleotide divergence of the ITS region between T. torulosum and T. flavidosporum is 90% (58/577‒586 bp), and that between T. torulosum and the T. turmericum-T. xanthomonosporum complex is 86‒88% (71‒80/577‒586 bp). In the LSU phylogeny, T. torulosum is placed within the Japonicum clade with T. japonicum and T. flavidosporum (100/1.00), which is placed in the basal group in the genus Tuber (Fig. 3).

Fig. 3 - A RAxML tree of Tuber torulosum and related taxa based on rDNA LSU sequences. ML bootstrap values and Bayesian posterior probabilities are indicated on branches as MLBS/BPP. Values supported by both BS ≥ 75% and PP ≥ 0.95 are shown.

The Japonicum clade is thought to be an early diverging lineage of the genus Tuber (Bonito et al., 2013), which was supported by our LSU phylogenetic tree (Fig. 3). Interestingly, all the members of this clade share two ascospore characters: one- or two-spored asci and pale yellow spherical spores. These characters are not shared by any other Tuber species. As expected based on our preliminary observations, phylogenetic analyses showed that T. torulosum belongs in the Japonicum clade (Fig. 2) and is more closely related to Japanese T. flavidosporum and the Chinese T. turmericum-T. xanthomonosporum complex than to T. japonicum. This is reflected by the number of spores in the ascus: T. japonicum generally forms two spores per ascus, whereas the former three species form monosporic asci. Therefore, the Japonicum clade is clearly divided into one- and two-spored groups.

The four one-spored species in the Japonicum clade (T. torulosum, T. flavidosporum, T. turmericum, and T. xanthomonosporum) are very similar morphologically, but several features distinguish the species. The tomentose mycelium covering the ascoma surface is observed only in T. torulosum. The reticulum of T. torulosum ascospores has fewer, wider meshes across the spore width (3 meshes, 15‒17 × 10‒15 µm) than those of T. flavidosporum (3 or 4 meshes, 11‒18 × 10‒15 µm) (Kinoshita et al., 2016), T. turmericum (4 or 5 meshes, < 10 µm wide according to Fig. 2 in Fan et al., 2015), and T. xanthomonosporum (4 or 5 meshes, 8‒15 × 7‒13 µm) (Qing et al., 2015). Therefore, both morphological and molecular analyses support that T. torulosum is new to science. Also, moniliform cystidia emerging from the tomentose mycelium is a diagnostic feature for T. torulosum, but similar cystidia have been observed on the peridium of all species in the Gibbosum clade (Bonito, Trappe, Rawlinson, & Vilgalys, 2010), as well as T. japonicum and T. flavidosporum (Kinoshita et al., 2016). Sbrana et al. (2000) reported that in pure culture the abundance of moniliform mycelia of Tuber borchii Vittad. increased with the presence of bacteria. Species in the Japonicum and Gibbosum clades both form similar ascoma that are smooth with furrows that are reddish-brown at maturity, implying that these truffles may be more susceptible to soil bacteria than black truffles, which are covered by a melanized peridium.

Tuber torulosum has only been found in one site in Japan. However, in the ITS phylogenetic analysis, the sequence of Q. liaotungensis ectomycorrhiza (KX444500, Wang, Liu, Long, Han, & Huang, 2017) fell into the same clade as T. torulosum, with only one nucleotide substitution difference (Fig. 2). The environmental sample was collected near the border between China and North Korea (40.47°N, 124.10°E), indicating that they are highly likely conspecific. This further suggests that T. torulosum has a wide distribution in East Asia, but splits into each population by isolating the Japanese archipelago from Eurasia after Riss glaciation in the late Pleistocene (ca. 0.15Ma). A similar distribution pattern has been shown for Asian black truffles, T. himalayense and T. longispinosum (Feng, Zhao, Xu, Qin, & Yang, 2016; Kinoshita et al., 2018; El Karkouri, Couderc, Decloquement, Abeille, & Raoult, 2019; Park et al., 2021). These data may help to clarify the process of diversification of Tuber in East Asia.

• Key to the Tuber species in the Japonicum group

The members of the Japonicum clade differ from those of other lineages in the genus Tuber by forming asci that contain one or two ascospores, which are globose and pale yellow with irregularly reticulate ornamentation.

• 1a. Ascospores two per ascus...... T. japonicum

• 1b. Ascospores one per ascus, rarely 2-spored...... 2

2a. Ascoma surface partially covered by tomentose mycelium, composed of moniliform cystidia, ascospore ornaments 3 meshes across ascospore diameter...... T. torulosum

2b. Ascoma surface smooth, cystidia scabrous, ascospore ornaments 3 or 4 meshes across ascospore diameter; Japan...... T. flavidosporum

2c. Ascoma surface smooth, cystidia spiky, ascospore ornaments coarsely 4 or 5 meshes across diameter; southern China...... T. turmericum-T. xanthomonosporum complex

Disclosures

The authors declare no conflict of interest. All the experiments undertaken in this study comply with the current laws of Japan.

Acknowledgments

We are grateful to Mika Tatara and Izumi Tsukiashi for helping with molecular and morphological analyses. We are grateful to two anonymous reviewers for their helpful comments that improved the manuscript. This work was supported by JSPS KAKENHI Grant number 19K06137.

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