Tuber elevatireticulatum sp. nov., a new species of whitish truffle from Taiwan

Abstract

Background

There are estimated 180–220 species of Tuber described in the world, but the diversity of the genus in Taiwan is poorly known, with only two species recorded, i.e., Tuber formosanum and T. furfuraceum. During our survey of hypogenous fungi in Taiwan, a whitish truffle belongs to Puberulum clade was collected from roots of Keteleeria fortunei var. cyclolepis in central Taiwan and appeared to differ from the two recorded species.

Results

The whitish truffle is herein described as a new species Tuber elevatireticulatum, which is distinguished from closely resembled Asian whitish truffles species like Tuber thailandicum, T. panzhihuanense, T. latisporum and T. sinopuberulum by the association with Keteleeria host, small light brown ascocarps with a dark brown gleba, dark brownish and elliptical ascospores ornamented with a prominently raised alveolate reticulum. Molecular phylogenetic analyses of both ITS and LSU loci clearly supports T. elevatireticulatum as a new species without any significant incongruence.

Conclusions

The whitish truffle is herein described as a new species T. elevatireticulatum based on the evidence from morphology and DNA sequences. T. elevatireticulatum is the first scientific record of whitish truffle in Taiwan.

Background

True truffles, belonging to the genus Tuber (Tuberaceae, Pezizales, Pezizomycetes), produce hypogeous ascocarps, which are formed in soil or sometimes within layers of leaf litter. They have lost the ability to actively discharge ascospores (Bonito and Smith 2016). They are symbiotic fungi that develop association with fine roots of specific host trees (T. oregonense Trappe, Bonito and P. Rawl. with Douglas fir) or broad host ranges (T. aestivum (Wulfen:Fr.) Spreng. with some plant species in Betulaceae, Corylaceae, Fagaceae, Tiliaceae, Pinaceae and Cistaceae) (Hall et al. 2007). The unique aroma makes some species greatly sought after as high-end culinary ingredients throughout the world, especially in Europe (Hall et al. 2007). The scarcity and irreplaceably scent of French Périgord black truffle (T. melanosporum Vittad.) and Italian Alba white truffle (T. magnatum Pico.) render them among the most famous and demanding truffles in the world (Hall et al. 2007; Bonito et al. 2010a).

Index Fungorum (http://www.indexfungorum.org/names/Names.asp) lists out three hundred and five Tuber names, however, many of them required clarification (Suwannarach et al. 2015; Kinoshita et al. 2016). Bonito et al. (2013) reassessed the published names and estimated 180–220 accepted species in the genus, was subdivided into 11 major clades according to their phylogenetic relationships. Puberulum clade, Maculatum clade and closely related lineage Gibbosum clade were phylogenetically grouped with as Puberulum Group and members of this group commonly called “whitish truffle” in order to distinguish them from Italian white truffle (T. magnatum in Aestivum clade) (Bonito et al. 2010a; Lancellotti et al. 2016). Researches in Tuber have a long history and are well-documented in Europe and North America. However, research in Asia are still scarce despite the estimated high diversity (Bonito et al. 2010a; Kinoshita et al. 2011). Hypogeous fungi in Taiwan are poorly documented, with only T. formosanum Hu (invalidly described in 1992 due to the lack of designated holotype and later re-typification in 2013) and T. furfuraceum Hu and Wang reported previously. Both species form symbiotic association with roots of Quercus glauca (Thunb. ex Murray) Oerst. in the family of Fagaceae (Hu 1992; Hu and Wang 2005; Qiao et al. 2013). A whitish truffle was mentioned in Hu (1987) but lacks a formal description.

During our survey of hypogenous fungi in Taiwan, a whitish truffle was found under Keteleeria fortunei var. cyclolepis (Flous) Silba, in Sitou Tract, Nantou County of central Taiwan. It resembles several known Asian whitish truffles in the Puberulum Clade, such as T. thailandicum Suwannarach et al. (2015), T. panzhihuanense Deng et al. (2013), T. latisporum Chen and Liu (2007), T. pseudosphaerosporum Fan and Yue (2013), and T. alboumbilicum Wang and Li (Li et al. 2014), but differs from species in the Puberulum clade by the only species associated with Keteleeria host, small light brown ascocarps with hyphae-like hairs arised, dark brownish and elliptical ascospores ornamented with a prominently raised alveolate reticulum.

Methods

Sample collection

Ascocarps were collected with three-pronged garden cultivators, wrapped with tissue paper and kept in separate plastic zipper bags until further morphological and molecular analyses in laboratory. Ascocarps were weighted freshly within 24 h, and the pH value of adjacent soil were measured by JENCO 6010M pH meter following protocol of the manufacturer.

Morphological analysis

Ascocarps were cleaned with dry toothbrush, and then cut into halves for observing gleba color or color change under air exposure. Sections of fresh tissue were made with a razor blade by hand, then mounted in 0.1% (w/v) cotton blue in lacto-phenol for describing morphological characteristics by a Leica DMLB light microscope. Ascospore dimensions, with the ornamentation excluded, were based on at least 100 randomly selected ascospores. The range of ascospore length to width ratio (Q), average Q with ± standard deviation (Q) was calculated, and number of meshes across the ascospore width was measured.

For scanning electron microscopy (SEM), ascospores from dried gleba were mounted onto SEM stubs with carbon double-sided tape (Nisshin EM CO., Ltd, Tokyo), coated with gold–palladium, then examined and photographed with a tabletop HITACHI TM3000 SEM. Holotype was deposited at Herbarium of Taiwan Forestry Research Institute, Taipei, Taiwan (Index Herbarium: TAIF).

Molecular analysis

DNA extraction

Approximately 9–14 mg of gleba tissue of fresh ascocarps were ground by plastic pestle with 800 µl of Lysis Buffer (Taiwan Advanced Nanotech Inc.; containing Guanidine salt, Tris buffer and surfactants) in 1.5 ml centrifuge tube for DNA extraction. DNA was then extracted using the TANBead^Ⓡ fungal Nucleic Acid Extraction Kit and TANBead^Ⓡ Nucleic Acid Extractor (Taiwan Advanced Nanotech Inc.) following protocol of the manufacturer.

Polymerase chain reaction (PCR) amplification and sequencing

Two nuclear ribosomal DNA loci were used for amplifying and sequencing, including the internal transcribed spacer (ITS) with forward primer ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) was paired with reverse primer ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) (White et al. 1990); and ribosomal large subunit (LSU) with forward primer LR0R (5′-ACCCGCTGAACTTAAGC-3′) (Rehner and Samuels 1994) was paired with reverse primer LR5 (5′-TCCTGAGGGAAACTTCG-3′) (Vilgalys and Hester 1990). PCR was performed in 25 µl reactions containing 2.5 µl DNA template, 1 µl primer each, 8 µl ddH₂0 and 12.5 µl 2× Taq Master Mix (including 20 mM KCl, 4 mM MgSO₄·7H₂O, 40 mM Tris–HCl with pH 8.8, 0.2% Triton X-100, 20 mM (NH₄)₂SO₄, 0.2 mg/ml BSA, 0.4 mM dNTP mix, 100 U/ml Taq DNA Polymerase and stabilizers) (Genomics Bioscience and Technology CO., Ltd.). PCR for ITS/LSU were run as an initial denaturation at 94/95 °C for 3/2 min, then at 94/95 °C for 30 s, annealing at 56/50 °C for 30 s, extension at 72 °C for 30 s/1 min by 30 cycles and a final extension at 72 °C for 5/10 min on a multigene thermal cycler (Labnet International, Inc.). PCR products were checked on agarose gel containing 1.4% agarose and 0.5× Tris–acetate-EDTA (TAE) and stained with 5 µl/100 ml Healthview™ nucleic acid stain under UV light by multilmage™ light cabinet (Alphalmager 2200). The PCR products were sent to Seeing Bioscience Co., Ltd. for purification and sequencing by Sanger Sequencing Method (ABI 3730).

Phylogenetic analyses

Six ITS and eight LSU sequences were obtained from ascocarps of T. elevatireticulatum and were submitted to GenBank with Accession Numbers MF540616–MF540621 (ITS) and LSU sequences: LC425119–LC425126 (LSU). Other whitish Tuber sequences were obtained from GenBank database for phylogenetic analyses (Table 1), with Choiromyces alveolatus as the outgroup. Sequences were aligned using MAFFT 7 (Katoh and Standley 2013) with default settings, and poorly aligned sites were identified using Gblocks 0.91b (Castresana 2000) with gaps allowed in conserved blocks and with all other parameters left as default values. Ambiguous sites were excluded from phylogenetic analyses. Maximum likelihood (ML) analyses were conducted with MEGA 6.0 (Tamura et al. 2013) using K2P model. Bootstrap analyses were conducted with 1000 replications (Felsenstein 1985). Bayesian phylogenetic analyses were conducted with MrBayes 3.2.6 (Ronquist et al. 2012), for evaluating the effect of different phylogenetic approach. K2P model was used and MCMC chains were run for 1,000,000 generations, sampling every 100th tree. Among these, the first 20% trees were discarded as burn-in phase and the remaining trees were used to calculate Bayesian posterior probabilities. The consensus tree was viewed with FigTree 1.4.3 (Rambaut 2014).

Table 1 Details of the whitish Tuber ITS sequences used in phylogenetic study

Results

Taxonomy

Tuber elevatireticulatum K.F. Wong and H.T. Li, sp. nov. Fig. 1

Fig. 1

Tuber elevatireticulatum. a Mature ascocarp. b, c Cross section of ascocarp showing a dark brown gleba with narrow, light brown veins. d Section of peridium and gleba. e Pseudoparenchymatous tissue of peridium. f Hyphae-like hairs arising from outermost cells. g Ascospores. h Scanning electron micrograph of an ascospore. Bars: a, b 3.5 mm; c 1.5 mm; d 500 µm; e–g 50 µm; h 10 µm

MycoBank no.: MB824068.

Etymology: Referring to the prominently elevated reticulum on the ascospores.

Ascocarp hypogeous, scattered, solitary, subglobose or irregular, 12–19 mm long × 10–15 mm wide, 0.32–1.7 g in fresh weight, solid, smooth on the surface, whitish to pale yellowish when fresh, becoming light brown at maturity. Peridium two-layered; inner layer 85–425 μm thick, hyaline, composed of intricately interwoven hyphae; outer layer 75–110 μm thick, light brownish, pseudoparenchymatous, composed of globose, subglobose, rod-shaped or angular cells, 5–25 μm diam. Hyphae-like hairs arise from outermost cells, hyaline, septate, tapering towards the ends, acute or round at the apex, 50–275 × 1.25–3.75 μm. Gleba translucent or light-brown, marbled with narrow, white veins when young, becoming dark brown, marbled with narrow, light brown veins at maturity. Asci 1-3(-4)-ascospored, globose, subglobose, ovoid to ellipsoid, 47.5–88 × 37.5–75 µm, hyaline, with a wall 2.5 µm thick. Ascospores broadly ellipsoid to ellipsoid, rarely subglobose and globose, with mature ascospore ratio ranging 0.2–53% (n = 1000), yellowish brown to dark brown, with a wall 2.5–5 µm thick, 32.5–50 × 20–32.5 µm from 1-ascospored asci, 20–48 × 20–32.5 µm from 2-ascospored asci, 20–40 × 20–27.5 µm from 3-ascospored asci, 22.5–35 × 17.5–25 µm from 4-ascospored asci (Q = 1.0–1.75, Q = 1.30 ± 0.19), ornamented with irregular reticulations 2.5–7.5 µm high, with meshes varying in size, mostly 3-4(-5) across the ascospore width.

Specimens examined: TAIWAN, Nantou County, Sitou Tract, associated with roots of K. fortunei var. cyclolepis, 1 Jun 2017, collected by C.-L. Lin, K.-F. Wong, H.-T. Li and F.-Y. Lin, XTAM3 (holotype), ITS sequences: MF540616–MF540621; LSU sequences: LC425119–LC425126.

Notes: Tuber elevatireticulatum grows in montane area of central Taiwan with elevation of 1150 m. It is associated with a cluster of K. fortunei var. cyclolepis in a mixed coniferous plantation, at least 4 m apart from the nearest Cryptomeria japonica (L. f.) D. Don, Chamaecyparis formosensis Matsum. and a few Pinus species which all have no record of association with Tuber species. Ascocarps are mostly scattered and distributed in solitary in loamy soil with pH ranging from 5 to 6. Ascocarps are usually found within 0–2 m from tree trunks, starting to develop in March and maturing in June. Odor is pleasant, mild, peculiar but superb, rarely becoming unpleasant with ageing. The temperature during the ascocarp formation is 20–25 °C.

Phylogenetic analyses

The ITS matrix consisted of 52 sequences and 1661 aligned bases, of which 1198 bp were identified as poorly aligned and were excluded by Gblocks. The resultant ITS alignment was 463 bp. The LSU matrix consisted of 47 sequences and 1519 aligned bases, of which poorly aligned and were excluded by Gblocks and the resultant LSU alignment was 580 bp. As Maximum likelihood and Bayesian analyses yielded similar tree topologies of ITS region, thus the only tree generated form ML analysis is shown in Fig. 2. The ML and Bayesian analyses of LSU region is similar in general, due to the limited availability of sequences in database, the tree inferred form ML analysis is presented in Fig. 3, separate trees are presented as Additional files 1, 2.

Fig. 2

Phylogenetic tree of Tuber elevatireticulatum and related whitish truffles based on the ITS-rDNA sequences. Choiromyces alveolatus was used as the outgroup taxa. Numbers identify the bootstrap values and Bayesian posterior probabilities are indicated near branches as BS/PP. Values of BS and PP below 50% are not indicated. The sequences of new species described in this study are bold as indication

Fig. 3

Phylogenetic tree of Tuber elevatireticulatum and related whitish truffles based on the LSU-rDNA sequences. Choiromyces alveolatus was used as the outgroup taxa. Numbers identify the bootstrap values and Bayesian posterior probabilities are indicated near branches as BS/PP. Values of BS and PP below 50% are not indicated. The sequences of new species described in this study are bold as indication

There has no significant incongruence among ITS and LSU region of ribosomal DNA. Tuber elevatireticulatum is clearly different from other whitish truffles and formed a monophyletic clade with strong bootstrap (BS) and posterior probability (PP) values (1.00/1.00). Based on the ITS analysis, T. elevatireticulatum was placed clearly in the Puberulum clade, within which it formed a subclade with five Asian species, including T. thailandicum, T. pseudosphaerosporum, T. alboumbilicum, T. latisporum, and T. panzhihuanense, with strong branching supports by BS (0.89) and PP (0.99) value. Also included in the Puberulum clade were T. borchii, T. dryophilum, T. oligospermum and T. sphaerospermum from Europe; T. microsphaerosporum, T. sinopuberulum, T. vesicoperidium, T. lijiangense, T. sinosphaerosporum, T. zhongdianense, T. huizeanum, T. liui and T. liyuanum from China; and T. californicum from the USA. These whitish truffle species formed a subclade within the Puberulum clade with strong PP value of 1.00 and was sister to the one where T. elevatireticulatum was placed. The groupings of whitish truffles were similar from those in Kinoshita et al. (2011), Suwannarach et al. (2015) and Bonito and Smith (2016).

Discussion

Tuber elevatireticulatum is distinguished from other whitish truffle species by the only species associated with Keteleeria host, its small light brown ascocarps with a dark brown gleba and brown, ellipsoid ascospores with a prominent raised alveolate reticulum. Phylogenetic analyses clearly placed T. elevatireticulatum among other whitish truffle species in the Puberulum clade as a distinct taxon. Morphologically, truffles belonging to the Puberulum clade tend to have small and light-colored ascocarps, globose to subglobose ascospores with an alveolate-reticulate ornamentation (Bonito and Smith 2016). However, ascospores of T. elevatireticulatum are mostly ellipsoid, resembling those of the species in the Maculatum clade.

Tuber elevatireticulatum clustered in a subclade of the Puberulum group with several Asian whitish truffle species, including T. thailandicum, T. pseudosphaerosporum, T. alboumbilicum, T. panzhihuanense, and T. latisporum (Fig. 2). Tuber elevatireticulatum is similar to T. thailandicum in having a dark brown gleba at maturity, hyphae-like hairs, and the size of alveolae of the reticulum. However, T. thailandicum differs by having a larger ascocarp size (> 2 cm in diam.), a thinner peridium (150–225 µm), shorter hyphae-like hairs (20–63.5 µm), subglobose ascospores with a smaller Q value (1.09 ± 0.08), and larger ascospores in one-ascospored asci (40–65 × 40–62 µm) (Suwannarach et al. 2015). In addition, T. thailandicum is associated with roots of Betula, whereas T. elevatireticulatum is with Keteleeria roots, a host previously unknown to Tuber species. Tuber elevatireticulatum resembles T. pseudosphaerosporum in having light-colored ascocarps with a smooth surface and the same numbers of ascospores in asci but differs from the latter by a smaller ascocarp size, well-developed hyphae-like hairs, larger ellipsoid ascospores, a lower reticulum, and occurrence in a different season (Fan and Yue 2013). Tuber alboumbilicum is different from T. elevatireticulatum by a smaller ascocarp size (< 1 cm), a thinner peridium, and globose ascospores. Tuber panzhihuanense is distinct from T. elevatireticulatum by a dark grey to blackish gleba (Deng et al. 2013). Tuber latisporum is different from T. elevatireticulatum by reddish brown ascocarps, a blackish gleba and larger ascospores (62–93 × 41–74 µm) (Chen and Liu 2007). Beyond this subclade, Tuber sinopuberulum resembles T. elevatireticulatum in having light brown ascocarps with a smooth surface but differs from it in lacking hyphae-like hairs arising from the peridium, a light brown to brown gleba color, and globose ascospores (Fan et al. 2012).

Truffles in general favor dry, alkaline and calcareous soil (Hall et al. 2007), but T. elevatireticulatum was found in an area with a subtropical humid climate, slightly acidic soil of pH 5–6, and relatively high annual rainfall. This phenomenon has also been observed in Asia like Japan (Kinoshita et al. 2011) and Thailand (Suwannarach et al. 2015).