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Two new truffle species, Tuber japonicum and Tuber flavidosporum spp. nov. found from Japan
m y c o s c i e n c e x x x ( 2 0 1 6 ) 1 e8
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Two new truffle species, Tuber japonicum and Tuber flavidosporum spp. nov. found from Japan Akihiko Kinoshita a,*, Hiromi Sasaki b, Kazuhide Nara c a Department of Forest Microbiology, Forestry and Forest Products Research Institute, Matsunosato, Tsukuba, Ibaraki, 305-8687, Japan b Mycologist Circle of Japan, Fujisawa, Kanagawa, 252-0804, Japan c Graduate School of Frontier Sciences, The University of Tokyo, Kashiwanoha, Kashiwa, Chiba, 277-8563, Japan
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abstract
Article history:
We describe and illustrate two new species, Tuber japonicum and Tuber flavidosporum, based
Received 8 March 2016
on molecular and morphological analyses. Tuber japonicum is characterized by its two-
Received in revised form
spored asci and pale yellow irregularly reticulate ascospores. Tuber flavidosporum has one
21 June 2016
ascospore per ascus, which is similar to some related species in China, but is distin-
Accepted 21 June 2016
guishable by its large reticulate ornamentation. Molecular phylogenetic analysis also
Available online xxx
supports T. japonicum and T. flavidosporum as new species. © 2016 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Keywords: Ascomycota Biogeography Ectomycorrhizal fungi Edible mushroom Tuberaceae
1.
Introduction
The genus Tuber F.H. Wigg. belongs to the Tuberaceae in Pezizales, Ascomycota. Fruit bodies of some Tuber species are known as “truffles”, which are highly prized due to their aromatic qualities. Two European species in particular, Tuber melanosporum Vittad. (Perigord black truffle) and Tuber magnatum Pico (Piedmont white truffle), are among the most famous truffles in the world. The genus is also known to form ectomycorrhizal associations and help the growth of major forest tree species such as the Betulaceae, the Fagaceae and
the Pinaceae (Zambonelli and Bonito 2013). Because of its economic and ecological importance, the taxonomy of Tuber has attracted much attention. Index Fungorum lists 290 species, subspecies and varieties, but these could potentially include many synonyms. A recent molecular phylogenetic study suggests that the genus comprises at least 180 species (Bonito et al. 2010). Tuber diversity is relatively well documented in Europe (Ceruti et al. 2003), North America (Trappe et al. 2009) and China (e.g., Garcı´a-Montero et al. 2010), but the species diversity in other regions is poorly understood. In Japan, the first public report on Tuber was released in 1976 (Trappe 1976), and described T. californicum
* Corresponding author. Tel.: þ81 29 829 8244; fax: þ81 29 873 1542. E-mail address: [email protected] (A. Kinoshita). http://dx.doi.org/10.1016/j.myc.2016.06.006 1340-3540/© 2016 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. Please cite this article in press as: Kinoshita A, et al., Two new truffle species, Tuber japonicum and Tuber flavidosporum spp. nov. found from Japan, Mycoscience (2016), http://dx.doi.org/10.1016/j.myc.2016.06.006
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Harkn. Later, a few local reports noted the records of European and North American species (e.g., T. aestivum (Wulfen) Spreng., T. californicum and T. magnatum; e.g., Yoshimi 2008) in Japan, although their taxonomical identity is uncertain. Recently, we have conducted molecular phylogenetic analyses for the 186 ascomata collections newly sampled in Japan from 1999 to 2008. This study demonstrated that the Japanese truffles include at least 20 species, most of which are different from European and North American species and likely to be new species (Kinoshita et al. 2011). Moreover, we found a new phylogroup, the Japonicum group, composed of only two Japanese species (Tuber sp. 8 and sp. 9) and distantly related to other known phylogroups. Common morphological characteristics of the phylogroup are whitish to pale yellow colored ascocarp, and one or two ascospores per ascus. Here, we formally describe these two Japanese species based on detailed morphological and molecular phylogenetic analyses.
2.
Materials and methods
2.1.
Morphological observations
Morphological observation was performed for the collections of fresh and dried ascocarp specimens used in our phylogenetic study (Kinoshita et al. 2011) and a few additional samples. For macroscopic characteristics, we observed ascocarp size, ornamentation, and colors following the Munsell system, mostly using fresh specimens. Microscopic features of fresh and dried specimens were observed in water, 3% (w/v) KOH solution, and Melzer's reagent. Photographs were taken under a light microscope and then we measured the size of fully matured ascospores and asci, peridium thickness, and other microscopic features using PhotoRuler 1.1 (http://hyogo. inocybe.info/_userdata/ruler/help-eng.html). For scanning electron microscopy (SEM), spores were scraped from the dried gleba and mounted in distilled water on a cover glass. After drying, the cover glass was pasted directly onto an SEM stub with double-sided tape, coated with gold-palladium, and photographed with a HITACHI S-4800. The examined specimens were deposited in the Mycological Herbarium of Forestry and Forest Products Research Institute Herbarium (TFM).
2.2.
Phylogenetic analyses
In the previous study, we demonstrated that the two Tuber species were phylogenetically distinct from the known species based on four nuclear loci (Kinoshita et al. 2011). To include recent sequence data of potentially related species described later (e.g., Fan et al. 2015) in our phylogenetic analyses, we conducted BLAST searches (blastn; Altschul et al. 1997) and retrieved related sequences from the International Nucleotide Sequence Database (INSD, DDBJ/EMBL/GenBank databases; Table 1). In the internal transcribed spacer (ITS) data set, we included all sequences for each of the two species and their closest match sequences found in the INSD. We aligned the ITS data set using MAFFT 7 (Katoh and Standley 2013) with default settings, and poorly aligned sites were identified using Gblocks 0.91B (Castresana 2000) with a minimum block-length parameter of 5 and with gaps allowed in
conserved blocks, with all other parameters left at default values. All identified ambiguous sites were excluded before phylogenetic analyses. Maximum likelihood (ML) analyses were conducted with PhyML 3.0 (Guindon et al. 2010) using a GTR þ G6 þ I model, which was selected by Smart Model Selection (SMS) implemented in PhyML to determine the best substitution model. The approximate likelihood ratio test (aLRT), SH-like fast likelihood-based optimization method was used to evaluate branching support. The ML tree was visualized with MEGA 6 (Tamura et al. 2013). To evaluate the effect of different phylogenetic approaches, we also conducted Bayesian phylogenetic analyses with MrBayes 3.2.1 (Ronquist et al. 2012). In the Bayesian analysis, after selecting the best substitution models as determined by SMS (GTR þ G þ I), we ran two independent MCMC chains, sampling every 100th tree until the standard deviation of the split frequency (ASDSF) became <0.01. The log files of MrBayes were further analyzed using Tracer 1.6 (Rambaut et al. 2014) to check the effective sample sizes, which were always >100, 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 1502 trees (751 trees from each run) were used to construct a 50% majority rule consensus tree and the consensus trees were visualized with FigTree 1.4 (Rambaut 2014). The ITS alignment file was deposited in TreeBASE (Accession No. 18938).
3.
Results
3.1.
Taxonomy
Tuber japonicum H. Sasaki, A. Kinosh. & Nara, sp. nov. Fig. 1. MycoBank no.: MB815829. Ascomata subglobose or lobed, firm, whitish to pale yellow, 10e40 mm in diam. Peridium smooth, two layers, the outer layer pseudoparenchymatous and composed of irregular cells, the inner layer composed of interwoven hyphae. Gleba whitish to pale yellow marbled with white sterile veins. Asci (1e) 2(e3)-spored, 75e133 47e106 mm, spindle-to obovate-shaped. Ascospores globose, whitish to yellow, 25e50 mm in diam excluding reticulate ornamentation. Type: JAPAN, Miyagi Prefecture, Sendai, under Pinus densiflora Siebold et Zucc. and Quercus serrata Murray trees, 6 Nov 2007, collected by Hiromi Sasaki and Kazuhide Nara, N88 (holotype, TFM: S16001). Paratype, TFM: S16002eS16011. DNA sequences: holotype AB553444 (ITS); paratype AB553433eAB553434, AB553436eAB553441, AB553443, AB553445 (ITS), AB553519 (LSU), AB553539 (TEF-1a), AB553559 (RPB2). Etymology: japonicum (Lat.), referring to the species' occurrence in Japan only (Japanese name “Honseiyoshoro” from Hon- ¼ Japan, the type locality, seiyoshoro ¼ Japanese name for the genus Tuber). Ascomata hypogeous, subglobose to irregular and lobed, whitish or pale straw yellow when young, brown to dark brown at maturity, 10e40 mm in diam. Odor garlicky. Peridium smooth, 240e280 mm thick, composed of two layers:
Please cite this article in press as: Kinoshita A, et al., Two new truffle species, Tuber japonicum and Tuber flavidosporum spp. nov. found from Japan, Mycoscience (2016), http://dx.doi.org/10.1016/j.myc.2016.06.006
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Table 1 e Genbank accession numbers of ITS sequences used in our phylogenetic analysis. Species Choiromyces alveolatus C. alveolatus C. meandriformis Tuber borchii T. borchii T. californicum T. californicum T. gibbosum T. gibbosum T. japonicum T. japonicum T. japonicum T. japonicum T. japonicum T. japonicum T. japonicum T. japonicum T. japonicum T. japonicum T. japonicum T. japonicum T. japonicum T. japonicum T. japonicum T. flavidosporum T. latisporum T. maculatum T. maculatum T. oregonense T. oregonense T. pseudomagnatum T. pseudosphaerosporum T. shearii T. sinosphaerosporum T. sinosphaerosporum T. turmericum T. turmericum T. turmericum T. turmericum T. turmericum T. turmericum T. turmericum T. turmericum T. turmericum T. xanthomonosporum T. xanthomonosporum T. xanthomonosporum
Origin
Voucher no.
Genbank accession no.
USA USA USA Italy Italy USA USA USA USA Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan China Italy Denmark USA USA China China USA China China China China China China China China China China China China China China
MES97 HS2886 GB285 GB14 GB62 JT22590 JT28058 JT26632 JT6555 (epitype) H1 K179 (paratype) K228 (paratype) K426 K427 (paratype) K449 (paratype) K450 (paratype) K453 (paratype) K457 (paratype) K458 (paratype) K460 K461 (paratype) N88 (holotype) N90 (paratype) KN3.4 K213 (holotype) HKAS 44315 (holotype) A15 TL5974 JT15112 JT30493 BJTC FAN163 (holotype) BJTC FAN250 (holotype) OSC51052 BJTC FAN135 (holotype) BJTC FAN136 BJTC FAN459 BJTC FAN471 BJTC FAN472 (paratype) BJTC FAN473 (holotype) BJTC FAN474 BJTC FAN475 BJTC FAN476 BJTC FAN477 BJTC FAN482 YAAS L3185 (holotype) YAAS L3186 YAAS L3187
HM485332 HM485333 HM485331 HM485344 HM485342 HM485351 HM485346 FJ809862 FJ809863 AB553432 AB553433 AB553434 AB553435 AB553436 AB553437 AB553438 AB553439 AB553440 AB553441 AB553442 AB553443 AB553444 AB553445 LC128048 AB553446 DQ898183 AM406673 AJ969627 FJ809881 JQ925647 JQ771192 KF744063 HM485389 JX092086 JX092087 KT758834 KT758835 KT758836 KT758837 KT758838 KT758839 KT758840 KT758841 KT758842 KJ162154 KJ162155 KJ162156
New species described in this study are indicated as bold.
the outer layer 100e180 mm thick and pseudoparenchymatous with scabrous or rarely spindle-shaped cystidia up to 160 mm in length and 2e6 mm in diam, composed of irregular or rectangular cells, 14e29 9e16 mm, pale yellow or hyaline; the inner layer 110e130 mm thick, of complex interwoven cells. Gleba whitish to light brown marbled with white sterile veins. Asci mostly 2-spored, occasionally 1- or 3-spored, 75e133 47e106 mm, broadly ellipsoid, spindle- to obovateshaped. Ascospores globose, whitish to pale yellow when young, yellow at maturity, 31e45 31e47 mm (1-spored, n ¼ 20), 27e43 27e42 mm (2-spored, n ¼ 126), 26e35 26e31 mm (3-spored, n ¼ 19); excluding the reticulate
ornaments of 2e5 mm in height, composed of irregularly shaped meshes, usually 4e6 across the spore width. Habitat and distribution: woodland or forest edges under Lithocarpus, Quercus in the Fagaceae, Abies, Pinus in the Pinaceae and Betula, Carpinus in the Betulaceae. Additional specimens examined (paratype): JAPAN, IBARAKI Prefecture: Tsukuba city, under Carpinus tschonoskii Maxim., Lithocarpus edulis (Makino) Nakai, P. densiflora, Quercus myrsinifolia Blume and Q. serrata, 8 Nov 2004, collected by H. Sasaki, K427 (TFM: S16002); MIE Prefecture: Inabe city, 8 Dec 2007, collected by Masahito Taniguchi, K179 (TFM: S16003);
Please cite this article in press as: Kinoshita A, et al., Two new truffle species, Tuber japonicum and Tuber flavidosporum spp. nov. found from Japan, Mycoscience (2016), http://dx.doi.org/10.1016/j.myc.2016.06.006
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Fig. 1 e Tuber japonicum (TFM: S16002, holotype). A: Ascocarp. B, C: Light micrographs of ascospores. D: peridium. E: hyphal like hairs arising from the outermost cells of peridium. F: Scanning electron micrograph of ascospores. Bars: A 1 cm; B, C, F 30 mm; D, E 50 mm.
the same locality, under P. densiflora and Q. serrata trees, 4 Nov 2007, collected by H. Sasaki, K228 (TFM: S16004); MIYAGI Prefecture: Sendai city; 19 Oct 2006, collected by Yoko Ando, K449 (TFM: S16005); the same locality, 12 Nov 2006, collected by H. Sasaki K450 (TFM: S16006); the same locality, under P. densiflora and Q. serrata, 6 Nov 2007, collected by H. Sasaki, K457 (TFM: S16007), K458 (TFM: S16008); 10 Oct 2007, collected by H. Sasaki, K453 (TFM: S16009); Sendai city, 6 Nov 2007, under P. densiflora and Q. serrata, collected by H. Sasaki, K461 (TFM: S16010); Sendai city, under C. tschonoskii and Q. serrata, 6 Nov 2007, collected by H. Sasaki and K. Nara, N90 (TFM: S16011). Tuber flavidosporum H. Sasaki, A. Kinosh. & Nara, sp. nov. Fig. 2. MycoBank no.: MB815830. Ascomata subglobose, light brown to brown, 20 mm in diam. Peridium smooth, two layers, the outer layer
pseudoparenchymatous and composed of irregular cells, the inner layer composed of interwoven hyphae. Gleba whitish to pale yellow. Asci 1(e2)-spored, 73e97 56e77 mm, obovate to broadly ellipsoid in shape. Ascospores globose, reticulate, light yellow, 32e45 mm in diam excluding reticulate ornaments. Type: JAPAN, Kanagawa Prefecture, Kawasaki, under Cedrus deodara (Roxb.) G. Don trees, 26 Nov 2006, collected by Hiromi Sasaki, K213 (holotype, TFM: S16012). DNA sequences: holotype AB553446 (ITS), AB553520 (LSU), AB553540 (TEF1a), AB553560 (RPB2). Etymology: flavidosporum (Lat.), referring to the pale yellowish spores (Japanese name “Usukiseiyoshoro” from Usuki- ¼ pale yellow color, seiyoshoro ¼ Japanese name for the genus Tuber). Ascomata subglobose, light brown (10YR 7/4) to brown (10YR 5/4), 20 mm in diam. Odor indistinct. Peridium smooth, 200e250 mm thick, composed of two layers: the outer layer 100e140 mm thick and pseudoparenchymatous with scabrous
Please cite this article in press as: Kinoshita A, et al., Two new truffle species, Tuber japonicum and Tuber flavidosporum spp. nov. found from Japan, Mycoscience (2016), http://dx.doi.org/10.1016/j.myc.2016.06.006
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Fig. 2 e Tuber flavidosporum (TFM: S16012, holotype). A: Ascocarp. B, C: Light micrographs of ascospores. D: hyphal like hairs arising from the outermost cells of peridium. E: peridium. F: Scanning electron micrograph of ascospore. Bars: A 1 cm; B, C, D, F 30 mm; E 50 mm. cystidia up to 75 mm long and 2e5 mm in diam, composed of irregular shaped cells, 14e21 9e15 mm, pale yellow or hyaline; the inner layer 100e150 mm thick, of complex interwoven cells. Gleba whitish to pale yellow (5Y8/4) marbled with white (9.5N) sterile veins. Asci mostly 1-spored, very rarely 2-spored, 70e97 53e80 mm (n ¼ 52), obovate to broadly ellipsoid. Ascospores light yellow, globose, 32e46 mm (n ¼ 50) in diam excluding reticulate ornaments of 6e11 mm in height, composed of mostly irregular hexagonal meshes 11e18 10e15 mm, 3e4 across the spore width. Habitat and distribution: only one specimen was found, under planted C. deodara tree in a park.
3.2.
Phylogenetic analysis
The ITS sequence matrix contained 49 sequences and 769 aligned bases, of which 363 bp were identified as poorly aligned by Gblocks. All poorly-aligned sites were excluded
before phylogenetic analyses. The resultant ITS alignment was 406 bp, with 176 variable and 147 parsimony informative sites. ML and Bayesian analyses yielded similar tree topologies, and only the tree inferred from the ML analysis is shown in Fig. 3. Phylogenetic analysis of ITS sequences revealed three wellsupported lineages within the Japonicum group, irrespective of the method used (ML and Bayesian analyses). Fifteen sequences of Tuber japonicum formed a monophyletic lineage, which was sister to all the other sequences within the Japonicum group, with moderate branching support by SH-aLRT (78) and Bayesian posterior probability (0.81). Tuber flavidosporum represented another lineage, which was placed sister to a Chinese lineage comprising Tuber turmericum L. Fan and Tuber xanthomonosporum Qing & Y. Wang, with strong branching support (94/1.00). Tuber turmericum and T. xanthomonosporum fell into a single lineage with strong branching support (99/1.00).
Please cite this article in press as: Kinoshita A, et al., Two new truffle species, Tuber japonicum and Tuber flavidosporum spp. nov. found from Japan, Mycoscience (2016), http://dx.doi.org/10.1016/j.myc.2016.06.006
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Fig. 3 e Maximum likelihood phylogeny of Tuber japonicum and Tuber flavidosporum and related taxa based on the rDNA ITS sequences. SH-aLRT values and Bayesian posterior probabilities are indicated below branches as SH-aLRT/PP. Branches supported by both SH-aLRT ≥ 75% and PP ≥ 0.95 are shown as thickened black lines. Values below SH-aLRT < 50% or PP < 0.50 are not indicated.
4.
Discussion
The similarities of ITS sequences of T. japonicum and T. flavidosporum to all described Tuber species are lower than 81.5% and 85.7%, respectively, even to those described recently in the Japonicum group (Fan et al. 2015). Moreover, T. japonicum and T. flavidosporum can be distinguished from other related
species by morphology alone. In the Japonicum group, T. japonicum is unique in the number of ascospores per ascus: it usually has two ascospores per ascus (Fig. 1), whereas all the other described species (T. flavidosporum, T. turmericum and T. xanthomonosporum) have one ascospore per ascus. Although the morphological characters of T. flavidosporum resemble those of Chinese T. turmericum and T. xanthomonosporum, the spore ornamentation was larger than those of the two Chinese
Please cite this article in press as: Kinoshita A, et al., Two new truffle species, Tuber japonicum and Tuber flavidosporum spp. nov. found from Japan, Mycoscience (2016), http://dx.doi.org/10.1016/j.myc.2016.06.006
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species (Fig. 2). However, we obtained only one specimen and need more specimens to fully understand the variation in morphological traits of this species. The available molecular and morphological information clearly shows that the two Japanese truffle species, T. japonicum and T. flavidosporum, should be described as new species. Our phylogenetic analysis also brought to light that Chinese T. xanthomonosporum belongs to the Japonicum group with a high branching support (Fig. 3). Qing et al. (2015) reported this species as a Paradoxa-like species due to its one ascospore per ascus, but they concluded that its phylogenetic position remained unclear because of low branching support. In our phylogenetic analysis, however, T. xanthomonosporum and T. turmericum fell into a single clade having few ITS variations (99.6e99.9% similarities) with strong branch support (Fig. 3). Moreover, their morphological descriptions are similar to each other (whitish to pale yellow ascocarp and one ascospore per ascus), implying that T. xanthomonosporum and T. turmericum are highly likely synonyms (Fan et al. 2015; Qing et al. 2015). The Japonicum group, which includes the two new species described here, was firstly recognized in the genus Tuber by Kinoshita et al. (2011), and thereafter supported by Bonito et al. (2013). Although we assumed that this group was endemic to Japan, a recent study has shown that it is also found in China (Fan et al. 2015; but the authors proposed “T. turmericum group”). Therefore, the species belonging to the Japonicum group may have a wider distribution within Asia than assumed previously. Bonito et al. (2013) conducted a global phylogeographical analysis based on four nuclear loci using all available sequences of Tuber species, and demonstrated that the genus has a strong geographical pattern because of its dependence on animals for spore dispersal. A much smaller-scale biogeographical pattern became obvious in the Japonicum group, in which Japanese species were clearly separated from Chinese species, as in other Tuber groups found in our previous study (Kinoshita et al. 2011). Geographical separation of the Japanese archipelago from Eurasia may have enabled many Tuber species to evolve in multiple endemic groups independently.
Disclosures The authors declare no conflict of interest. All the experiments undertaken in this study comply with the current laws of Japan.
Acknowledgments We thank Yoko Ando, Hideko Miwa, Masaru Okubo, Haruko Saiki, Nanako Sato, Masahito Taniguchi for collecting samples, and Dr. Takashi Yamanaka, Dr. Jun-ichi Peter Abe, and Dr. Keisuke Obase for useful suggestions regarding this study. We also thank National Museum of Nature and Science, Tsukuba, Japan. We are grateful to Dr. Li Fan who provided the ITS sequences of T. turmericum and Dr. Katsushi Kuroda for helping with the use of scanning electron microscopy. This work was partly supported by JSPS KAKENHI Grants to AK
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(24710271), KN (21658054, 22380083, 15H02449), and also by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan, entitled “Technology development for the optimal use of forest resources”.
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Please cite this article in press as: Kinoshita A, et al., Two new truffle species, Tuber japonicum and Tuber flavidosporum spp. nov. found from Japan, Mycoscience (2016), http://dx.doi.org/10.1016/j.myc.2016.06.006
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journal homepage: www.elsevier.com/locate/myc
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Two new truffle species, Tuber japonicum and Tuber flavidosporum spp. nov. found from Japan Akihiko Kinoshita a,*, Hiromi Sasaki b, Kazuhide Nara c a Department of Forest Microbiology, Forestry and Forest Products Research Institute, Matsunosato, Tsukuba, Ibaraki, 305-8687, Japan b Mycologist Circle of Japan, Fujisawa, Kanagawa, 252-0804, Japan c Graduate School of Frontier Sciences, The University of Tokyo, Kashiwanoha, Kashiwa, Chiba, 277-8563, Japan
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abstract
Article history:
We describe and illustrate two new species, Tuber japonicum and Tuber flavidosporum, based
Received 8 March 2016
on molecular and morphological analyses. Tuber japonicum is characterized by its two-
Received in revised form
spored asci and pale yellow irregularly reticulate ascospores. Tuber flavidosporum has one
21 June 2016
ascospore per ascus, which is similar to some related species in China, but is distin-
Accepted 21 June 2016
guishable by its large reticulate ornamentation. Molecular phylogenetic analysis also
Available online xxx
supports T. japonicum and T. flavidosporum as new species. © 2016 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Keywords: Ascomycota Biogeography Ectomycorrhizal fungi Edible mushroom Tuberaceae
1.
Introduction
The genus Tuber F.H. Wigg. belongs to the Tuberaceae in Pezizales, Ascomycota. Fruit bodies of some Tuber species are known as “truffles”, which are highly prized due to their aromatic qualities. Two European species in particular, Tuber melanosporum Vittad. (Perigord black truffle) and Tuber magnatum Pico (Piedmont white truffle), are among the most famous truffles in the world. The genus is also known to form ectomycorrhizal associations and help the growth of major forest tree species such as the Betulaceae, the Fagaceae and
the Pinaceae (Zambonelli and Bonito 2013). Because of its economic and ecological importance, the taxonomy of Tuber has attracted much attention. Index Fungorum lists 290 species, subspecies and varieties, but these could potentially include many synonyms. A recent molecular phylogenetic study suggests that the genus comprises at least 180 species (Bonito et al. 2010). Tuber diversity is relatively well documented in Europe (Ceruti et al. 2003), North America (Trappe et al. 2009) and China (e.g., Garcı´a-Montero et al. 2010), but the species diversity in other regions is poorly understood. In Japan, the first public report on Tuber was released in 1976 (Trappe 1976), and described T. californicum
* Corresponding author. Tel.: þ81 29 829 8244; fax: þ81 29 873 1542. E-mail address: [email protected] (A. Kinoshita). http://dx.doi.org/10.1016/j.myc.2016.06.006 1340-3540/© 2016 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. Please cite this article in press as: Kinoshita A, et al., Two new truffle species, Tuber japonicum and Tuber flavidosporum spp. nov. found from Japan, Mycoscience (2016), http://dx.doi.org/10.1016/j.myc.2016.06.006
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Harkn. Later, a few local reports noted the records of European and North American species (e.g., T. aestivum (Wulfen) Spreng., T. californicum and T. magnatum; e.g., Yoshimi 2008) in Japan, although their taxonomical identity is uncertain. Recently, we have conducted molecular phylogenetic analyses for the 186 ascomata collections newly sampled in Japan from 1999 to 2008. This study demonstrated that the Japanese truffles include at least 20 species, most of which are different from European and North American species and likely to be new species (Kinoshita et al. 2011). Moreover, we found a new phylogroup, the Japonicum group, composed of only two Japanese species (Tuber sp. 8 and sp. 9) and distantly related to other known phylogroups. Common morphological characteristics of the phylogroup are whitish to pale yellow colored ascocarp, and one or two ascospores per ascus. Here, we formally describe these two Japanese species based on detailed morphological and molecular phylogenetic analyses.
2.
Materials and methods
2.1.
Morphological observations
Morphological observation was performed for the collections of fresh and dried ascocarp specimens used in our phylogenetic study (Kinoshita et al. 2011) and a few additional samples. For macroscopic characteristics, we observed ascocarp size, ornamentation, and colors following the Munsell system, mostly using fresh specimens. Microscopic features of fresh and dried specimens were observed in water, 3% (w/v) KOH solution, and Melzer's reagent. Photographs were taken under a light microscope and then we measured the size of fully matured ascospores and asci, peridium thickness, and other microscopic features using PhotoRuler 1.1 (http://hyogo. inocybe.info/_userdata/ruler/help-eng.html). For scanning electron microscopy (SEM), spores were scraped from the dried gleba and mounted in distilled water on a cover glass. After drying, the cover glass was pasted directly onto an SEM stub with double-sided tape, coated with gold-palladium, and photographed with a HITACHI S-4800. The examined specimens were deposited in the Mycological Herbarium of Forestry and Forest Products Research Institute Herbarium (TFM).
2.2.
Phylogenetic analyses
In the previous study, we demonstrated that the two Tuber species were phylogenetically distinct from the known species based on four nuclear loci (Kinoshita et al. 2011). To include recent sequence data of potentially related species described later (e.g., Fan et al. 2015) in our phylogenetic analyses, we conducted BLAST searches (blastn; Altschul et al. 1997) and retrieved related sequences from the International Nucleotide Sequence Database (INSD, DDBJ/EMBL/GenBank databases; Table 1). In the internal transcribed spacer (ITS) data set, we included all sequences for each of the two species and their closest match sequences found in the INSD. We aligned the ITS data set using MAFFT 7 (Katoh and Standley 2013) with default settings, and poorly aligned sites were identified using Gblocks 0.91B (Castresana 2000) with a minimum block-length parameter of 5 and with gaps allowed in
conserved blocks, with all other parameters left at default values. All identified ambiguous sites were excluded before phylogenetic analyses. Maximum likelihood (ML) analyses were conducted with PhyML 3.0 (Guindon et al. 2010) using a GTR þ G6 þ I model, which was selected by Smart Model Selection (SMS) implemented in PhyML to determine the best substitution model. The approximate likelihood ratio test (aLRT), SH-like fast likelihood-based optimization method was used to evaluate branching support. The ML tree was visualized with MEGA 6 (Tamura et al. 2013). To evaluate the effect of different phylogenetic approaches, we also conducted Bayesian phylogenetic analyses with MrBayes 3.2.1 (Ronquist et al. 2012). In the Bayesian analysis, after selecting the best substitution models as determined by SMS (GTR þ G þ I), we ran two independent MCMC chains, sampling every 100th tree until the standard deviation of the split frequency (ASDSF) became <0.01. The log files of MrBayes were further analyzed using Tracer 1.6 (Rambaut et al. 2014) to check the effective sample sizes, which were always >100, 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 1502 trees (751 trees from each run) were used to construct a 50% majority rule consensus tree and the consensus trees were visualized with FigTree 1.4 (Rambaut 2014). The ITS alignment file was deposited in TreeBASE (Accession No. 18938).
3.
Results
3.1.
Taxonomy
Tuber japonicum H. Sasaki, A. Kinosh. & Nara, sp. nov. Fig. 1. MycoBank no.: MB815829. Ascomata subglobose or lobed, firm, whitish to pale yellow, 10e40 mm in diam. Peridium smooth, two layers, the outer layer pseudoparenchymatous and composed of irregular cells, the inner layer composed of interwoven hyphae. Gleba whitish to pale yellow marbled with white sterile veins. Asci (1e) 2(e3)-spored, 75e133 47e106 mm, spindle-to obovate-shaped. Ascospores globose, whitish to yellow, 25e50 mm in diam excluding reticulate ornamentation. Type: JAPAN, Miyagi Prefecture, Sendai, under Pinus densiflora Siebold et Zucc. and Quercus serrata Murray trees, 6 Nov 2007, collected by Hiromi Sasaki and Kazuhide Nara, N88 (holotype, TFM: S16001). Paratype, TFM: S16002eS16011. DNA sequences: holotype AB553444 (ITS); paratype AB553433eAB553434, AB553436eAB553441, AB553443, AB553445 (ITS), AB553519 (LSU), AB553539 (TEF-1a), AB553559 (RPB2). Etymology: japonicum (Lat.), referring to the species' occurrence in Japan only (Japanese name “Honseiyoshoro” from Hon- ¼ Japan, the type locality, seiyoshoro ¼ Japanese name for the genus Tuber). Ascomata hypogeous, subglobose to irregular and lobed, whitish or pale straw yellow when young, brown to dark brown at maturity, 10e40 mm in diam. Odor garlicky. Peridium smooth, 240e280 mm thick, composed of two layers:
Please cite this article in press as: Kinoshita A, et al., Two new truffle species, Tuber japonicum and Tuber flavidosporum spp. nov. found from Japan, Mycoscience (2016), http://dx.doi.org/10.1016/j.myc.2016.06.006
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Table 1 e Genbank accession numbers of ITS sequences used in our phylogenetic analysis. Species Choiromyces alveolatus C. alveolatus C. meandriformis Tuber borchii T. borchii T. californicum T. californicum T. gibbosum T. gibbosum T. japonicum T. japonicum T. japonicum T. japonicum T. japonicum T. japonicum T. japonicum T. japonicum T. japonicum T. japonicum T. japonicum T. japonicum T. japonicum T. japonicum T. japonicum T. flavidosporum T. latisporum T. maculatum T. maculatum T. oregonense T. oregonense T. pseudomagnatum T. pseudosphaerosporum T. shearii T. sinosphaerosporum T. sinosphaerosporum T. turmericum T. turmericum T. turmericum T. turmericum T. turmericum T. turmericum T. turmericum T. turmericum T. turmericum T. xanthomonosporum T. xanthomonosporum T. xanthomonosporum
Origin
Voucher no.
Genbank accession no.
USA USA USA Italy Italy USA USA USA USA Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan Japan China Italy Denmark USA USA China China USA China China China China China China China China China China China China China China
MES97 HS2886 GB285 GB14 GB62 JT22590 JT28058 JT26632 JT6555 (epitype) H1 K179 (paratype) K228 (paratype) K426 K427 (paratype) K449 (paratype) K450 (paratype) K453 (paratype) K457 (paratype) K458 (paratype) K460 K461 (paratype) N88 (holotype) N90 (paratype) KN3.4 K213 (holotype) HKAS 44315 (holotype) A15 TL5974 JT15112 JT30493 BJTC FAN163 (holotype) BJTC FAN250 (holotype) OSC51052 BJTC FAN135 (holotype) BJTC FAN136 BJTC FAN459 BJTC FAN471 BJTC FAN472 (paratype) BJTC FAN473 (holotype) BJTC FAN474 BJTC FAN475 BJTC FAN476 BJTC FAN477 BJTC FAN482 YAAS L3185 (holotype) YAAS L3186 YAAS L3187
HM485332 HM485333 HM485331 HM485344 HM485342 HM485351 HM485346 FJ809862 FJ809863 AB553432 AB553433 AB553434 AB553435 AB553436 AB553437 AB553438 AB553439 AB553440 AB553441 AB553442 AB553443 AB553444 AB553445 LC128048 AB553446 DQ898183 AM406673 AJ969627 FJ809881 JQ925647 JQ771192 KF744063 HM485389 JX092086 JX092087 KT758834 KT758835 KT758836 KT758837 KT758838 KT758839 KT758840 KT758841 KT758842 KJ162154 KJ162155 KJ162156
New species described in this study are indicated as bold.
the outer layer 100e180 mm thick and pseudoparenchymatous with scabrous or rarely spindle-shaped cystidia up to 160 mm in length and 2e6 mm in diam, composed of irregular or rectangular cells, 14e29 9e16 mm, pale yellow or hyaline; the inner layer 110e130 mm thick, of complex interwoven cells. Gleba whitish to light brown marbled with white sterile veins. Asci mostly 2-spored, occasionally 1- or 3-spored, 75e133 47e106 mm, broadly ellipsoid, spindle- to obovateshaped. Ascospores globose, whitish to pale yellow when young, yellow at maturity, 31e45 31e47 mm (1-spored, n ¼ 20), 27e43 27e42 mm (2-spored, n ¼ 126), 26e35 26e31 mm (3-spored, n ¼ 19); excluding the reticulate
ornaments of 2e5 mm in height, composed of irregularly shaped meshes, usually 4e6 across the spore width. Habitat and distribution: woodland or forest edges under Lithocarpus, Quercus in the Fagaceae, Abies, Pinus in the Pinaceae and Betula, Carpinus in the Betulaceae. Additional specimens examined (paratype): JAPAN, IBARAKI Prefecture: Tsukuba city, under Carpinus tschonoskii Maxim., Lithocarpus edulis (Makino) Nakai, P. densiflora, Quercus myrsinifolia Blume and Q. serrata, 8 Nov 2004, collected by H. Sasaki, K427 (TFM: S16002); MIE Prefecture: Inabe city, 8 Dec 2007, collected by Masahito Taniguchi, K179 (TFM: S16003);
Please cite this article in press as: Kinoshita A, et al., Two new truffle species, Tuber japonicum and Tuber flavidosporum spp. nov. found from Japan, Mycoscience (2016), http://dx.doi.org/10.1016/j.myc.2016.06.006
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Fig. 1 e Tuber japonicum (TFM: S16002, holotype). A: Ascocarp. B, C: Light micrographs of ascospores. D: peridium. E: hyphal like hairs arising from the outermost cells of peridium. F: Scanning electron micrograph of ascospores. Bars: A 1 cm; B, C, F 30 mm; D, E 50 mm.
the same locality, under P. densiflora and Q. serrata trees, 4 Nov 2007, collected by H. Sasaki, K228 (TFM: S16004); MIYAGI Prefecture: Sendai city; 19 Oct 2006, collected by Yoko Ando, K449 (TFM: S16005); the same locality, 12 Nov 2006, collected by H. Sasaki K450 (TFM: S16006); the same locality, under P. densiflora and Q. serrata, 6 Nov 2007, collected by H. Sasaki, K457 (TFM: S16007), K458 (TFM: S16008); 10 Oct 2007, collected by H. Sasaki, K453 (TFM: S16009); Sendai city, 6 Nov 2007, under P. densiflora and Q. serrata, collected by H. Sasaki, K461 (TFM: S16010); Sendai city, under C. tschonoskii and Q. serrata, 6 Nov 2007, collected by H. Sasaki and K. Nara, N90 (TFM: S16011). Tuber flavidosporum H. Sasaki, A. Kinosh. & Nara, sp. nov. Fig. 2. MycoBank no.: MB815830. Ascomata subglobose, light brown to brown, 20 mm in diam. Peridium smooth, two layers, the outer layer
pseudoparenchymatous and composed of irregular cells, the inner layer composed of interwoven hyphae. Gleba whitish to pale yellow. Asci 1(e2)-spored, 73e97 56e77 mm, obovate to broadly ellipsoid in shape. Ascospores globose, reticulate, light yellow, 32e45 mm in diam excluding reticulate ornaments. Type: JAPAN, Kanagawa Prefecture, Kawasaki, under Cedrus deodara (Roxb.) G. Don trees, 26 Nov 2006, collected by Hiromi Sasaki, K213 (holotype, TFM: S16012). DNA sequences: holotype AB553446 (ITS), AB553520 (LSU), AB553540 (TEF1a), AB553560 (RPB2). Etymology: flavidosporum (Lat.), referring to the pale yellowish spores (Japanese name “Usukiseiyoshoro” from Usuki- ¼ pale yellow color, seiyoshoro ¼ Japanese name for the genus Tuber). Ascomata subglobose, light brown (10YR 7/4) to brown (10YR 5/4), 20 mm in diam. Odor indistinct. Peridium smooth, 200e250 mm thick, composed of two layers: the outer layer 100e140 mm thick and pseudoparenchymatous with scabrous
Please cite this article in press as: Kinoshita A, et al., Two new truffle species, Tuber japonicum and Tuber flavidosporum spp. nov. found from Japan, Mycoscience (2016), http://dx.doi.org/10.1016/j.myc.2016.06.006
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Fig. 2 e Tuber flavidosporum (TFM: S16012, holotype). A: Ascocarp. B, C: Light micrographs of ascospores. D: hyphal like hairs arising from the outermost cells of peridium. E: peridium. F: Scanning electron micrograph of ascospore. Bars: A 1 cm; B, C, D, F 30 mm; E 50 mm. cystidia up to 75 mm long and 2e5 mm in diam, composed of irregular shaped cells, 14e21 9e15 mm, pale yellow or hyaline; the inner layer 100e150 mm thick, of complex interwoven cells. Gleba whitish to pale yellow (5Y8/4) marbled with white (9.5N) sterile veins. Asci mostly 1-spored, very rarely 2-spored, 70e97 53e80 mm (n ¼ 52), obovate to broadly ellipsoid. Ascospores light yellow, globose, 32e46 mm (n ¼ 50) in diam excluding reticulate ornaments of 6e11 mm in height, composed of mostly irregular hexagonal meshes 11e18 10e15 mm, 3e4 across the spore width. Habitat and distribution: only one specimen was found, under planted C. deodara tree in a park.
3.2.
Phylogenetic analysis
The ITS sequence matrix contained 49 sequences and 769 aligned bases, of which 363 bp were identified as poorly aligned by Gblocks. All poorly-aligned sites were excluded
before phylogenetic analyses. The resultant ITS alignment was 406 bp, with 176 variable and 147 parsimony informative sites. ML and Bayesian analyses yielded similar tree topologies, and only the tree inferred from the ML analysis is shown in Fig. 3. Phylogenetic analysis of ITS sequences revealed three wellsupported lineages within the Japonicum group, irrespective of the method used (ML and Bayesian analyses). Fifteen sequences of Tuber japonicum formed a monophyletic lineage, which was sister to all the other sequences within the Japonicum group, with moderate branching support by SH-aLRT (78) and Bayesian posterior probability (0.81). Tuber flavidosporum represented another lineage, which was placed sister to a Chinese lineage comprising Tuber turmericum L. Fan and Tuber xanthomonosporum Qing & Y. Wang, with strong branching support (94/1.00). Tuber turmericum and T. xanthomonosporum fell into a single lineage with strong branching support (99/1.00).
Please cite this article in press as: Kinoshita A, et al., Two new truffle species, Tuber japonicum and Tuber flavidosporum spp. nov. found from Japan, Mycoscience (2016), http://dx.doi.org/10.1016/j.myc.2016.06.006
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Fig. 3 e Maximum likelihood phylogeny of Tuber japonicum and Tuber flavidosporum and related taxa based on the rDNA ITS sequences. SH-aLRT values and Bayesian posterior probabilities are indicated below branches as SH-aLRT/PP. Branches supported by both SH-aLRT ≥ 75% and PP ≥ 0.95 are shown as thickened black lines. Values below SH-aLRT < 50% or PP < 0.50 are not indicated.
4.
Discussion
The similarities of ITS sequences of T. japonicum and T. flavidosporum to all described Tuber species are lower than 81.5% and 85.7%, respectively, even to those described recently in the Japonicum group (Fan et al. 2015). Moreover, T. japonicum and T. flavidosporum can be distinguished from other related
species by morphology alone. In the Japonicum group, T. japonicum is unique in the number of ascospores per ascus: it usually has two ascospores per ascus (Fig. 1), whereas all the other described species (T. flavidosporum, T. turmericum and T. xanthomonosporum) have one ascospore per ascus. Although the morphological characters of T. flavidosporum resemble those of Chinese T. turmericum and T. xanthomonosporum, the spore ornamentation was larger than those of the two Chinese
Please cite this article in press as: Kinoshita A, et al., Two new truffle species, Tuber japonicum and Tuber flavidosporum spp. nov. found from Japan, Mycoscience (2016), http://dx.doi.org/10.1016/j.myc.2016.06.006
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species (Fig. 2). However, we obtained only one specimen and need more specimens to fully understand the variation in morphological traits of this species. The available molecular and morphological information clearly shows that the two Japanese truffle species, T. japonicum and T. flavidosporum, should be described as new species. Our phylogenetic analysis also brought to light that Chinese T. xanthomonosporum belongs to the Japonicum group with a high branching support (Fig. 3). Qing et al. (2015) reported this species as a Paradoxa-like species due to its one ascospore per ascus, but they concluded that its phylogenetic position remained unclear because of low branching support. In our phylogenetic analysis, however, T. xanthomonosporum and T. turmericum fell into a single clade having few ITS variations (99.6e99.9% similarities) with strong branch support (Fig. 3). Moreover, their morphological descriptions are similar to each other (whitish to pale yellow ascocarp and one ascospore per ascus), implying that T. xanthomonosporum and T. turmericum are highly likely synonyms (Fan et al. 2015; Qing et al. 2015). The Japonicum group, which includes the two new species described here, was firstly recognized in the genus Tuber by Kinoshita et al. (2011), and thereafter supported by Bonito et al. (2013). Although we assumed that this group was endemic to Japan, a recent study has shown that it is also found in China (Fan et al. 2015; but the authors proposed “T. turmericum group”). Therefore, the species belonging to the Japonicum group may have a wider distribution within Asia than assumed previously. Bonito et al. (2013) conducted a global phylogeographical analysis based on four nuclear loci using all available sequences of Tuber species, and demonstrated that the genus has a strong geographical pattern because of its dependence on animals for spore dispersal. A much smaller-scale biogeographical pattern became obvious in the Japonicum group, in which Japanese species were clearly separated from Chinese species, as in other Tuber groups found in our previous study (Kinoshita et al. 2011). Geographical separation of the Japanese archipelago from Eurasia may have enabled many Tuber species to evolve in multiple endemic groups independently.
Disclosures The authors declare no conflict of interest. All the experiments undertaken in this study comply with the current laws of Japan.
Acknowledgments We thank Yoko Ando, Hideko Miwa, Masaru Okubo, Haruko Saiki, Nanako Sato, Masahito Taniguchi for collecting samples, and Dr. Takashi Yamanaka, Dr. Jun-ichi Peter Abe, and Dr. Keisuke Obase for useful suggestions regarding this study. We also thank National Museum of Nature and Science, Tsukuba, Japan. We are grateful to Dr. Li Fan who provided the ITS sequences of T. turmericum and Dr. Katsushi Kuroda for helping with the use of scanning electron microscopy. This work was partly supported by JSPS KAKENHI Grants to AK
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(24710271), KN (21658054, 22380083, 15H02449), and also by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan, entitled “Technology development for the optimal use of forest resources”.
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Please cite this article in press as: Kinoshita A, et al., Two new truffle species, Tuber japonicum and Tuber flavidosporum spp. nov. found from Japan, Mycoscience (2016), http://dx.doi.org/10.1016/j.myc.2016.06.006