Tuber longispinosum A. Kinosh., 2018

Kinoshita, Akihiko, Nara, Kazuhide, Sasaki, Hiromi, Feng, Bang, Obase, Keisuke, Yang, Zhu L. & Yamanaka, Takashi, 2018, Using mating-type loci to improve taxonomy of the Tuber indicum complex, and discovery of a new species, T. longispinosum, PLoS ONE (e 0193745) 13 (3), pp. 1-21 : 9-15

Tuber longispinosum A. Kinosh. sp. nov. ( Fig 6A–6F ). [urn:lsid:mycobank.org:names: MB 821786].

Diagnosis: Differing from T. indicum and T. himalayense in ascospore ornamentation consisting of spines that are unconnected, and narrow at the base; and its significantly long spines.

Holotype: JAPAN, OITA Prefecture, under Quercus acutissima Carruth. , 7 Oct 2006, collected by Hiromi Sasaki, K447 ( TFM: S17009 ).

Ascomata subglobose, brown to dark greyish, 20–70 mm in diam. Peridium warty, two layers, the outer layer pseudoparenchymatous and composed of irregular cells. Gleba whitish

when young, becoming grayish brown to blackish at maturity with yellowish cream to white veins. Asci 1–5(–6)-spored, 59–72 × 50–59 μm, subglobose to broadly ellipsoid. Ascospores ellipsoid to subglobose with spiny ornamentation, brown to dark brown, (15–)21–35(–41) × (12–)15–26(–30) μm in diam excluding ornamentation.

Etymology: longispinosum (Lat.) , referring to “with long spine” from its spore ornamentation (Japanese name “ Iboseiyoshoro ” from ibo = warts, seiyoshoro = Japanese name for the genus Tuber ).

Ascomata: hypogeous, 20–70 mm in diam., subglobose and slightly lobed, firm, brown (10 R 4/8) to dark greyish (7.5YR 8/18), with low polygonal warts, 3–7 ridges, up to 900 μm high. Odor: aromatic, similar to seaweed or laver boiled in soy sauce when mature. Peridium: 400–800 μm thick, variable, pseudoparenchymatous, composed of two layers: outer layer 100– 200 μm thick, composed of irregular or ellipsoidal cells, 7–28 × 5–18 μm, with thick dark brown walls of 1–2 μm; inner layer 200–600 μm thick, composed of hyaline to yellowish, polygonal cells 5–15 × 5–10 μm that merge with glebal tissue of interwoven hyphae. Gleba: solid, whitish when young, becoming greyish brown to blackish at maturity, marbled with distinct, yellowish cream to whitish, meandering veins that merge at many points. Glebal tissue of interwoven hyphae: 3–8 μm broad with scattered cells, gelatinized, inflated up to 10 μm. Asci:

https://doi.org/10.1371/journal.pone.0193745.g005

typically subglobose to broadly ellipsoid, occasionally ellipsoid, variable depending on the number of spores, 59–72 × 50–59 μm (n = 177), rarely stipitate, 1–5(–6)-spored. Ascospore: ellipsoid, whitish or hyaline (glass-like) when young, becoming light brown (5 Y 8/3) to dark brown (5YR 9/4) at maturity, 31–41 × 22–30 μm, Q = 1.3–1.6 (1-spored, n = 18); 21–38 × 16– 29 μm, Q = 1.0–1.8 (2-spored, n = 78); 19–34 × 15–26 μm, Q = 1.1–1.7 (3-spored, n = 70); 15– 33 × 13–22 μm, Q = 1.0–2.1 (4-spored, n = 146); 16–31 × 12–20 μm, Q = 1.1–1.8 (5-spored,

n = 118); 15–26 × 13–18 μm, Q = 1.0–1.8 (6-spored, n = 24), excluding ornamentation, typically free spines, 3–7 (–12) μm (n = 430) in height with 1–4-μm bases.

Habitat and distribution: The fruiting period is from July to January. Woodland or forest edges under Abies ( Pinaceae ), Carpinus ( Betulaceae ), and Castanopsis and Quercus ( Fagaceae ).

Additional specimens examined (paratypes): JAPAN, KANAGAWA Prefecture, Atsugi City, under Carpinus tschonoskii Maxim. and Quercus serrata Murray , 9 Dec 2007, collected by Hiromi Sasaki, K 230 ( TFM: S 17001); Hayama-cho, under Castanopsis sieboldii (Makino) Hatsushima ex Yamazaki et Masiba and Quercus glauca Thunb. , 30 Dec 2002, collected by Hiromi Sasaki, K 70 ( TFM: S 17002); Ibid., 25 Oct 2004, collected by Kazuhide Nara, N 52 ( TFM: S 17004); Ibid., 23 Dec 2005, collected by Hiromi Sasaki, K 466 ( TFM: S 17003); SHIZUOKA Prefecture, Izu City, under Abies firma Sieb. Et Zucc. , Quercus salicina Blume , and Q. serrata , 31 Jan 2003, collected by Hiromi Sasaki, K 395 ( TFM: S 17005); KOCHI Prefecture, Umaji Village, under Q. glauca and Q. serrata , 3 Feb 2017, S 71 ( TFM: S 17006); EHIME Prefecture, Matsuyama City, 14 Oct 2006, collected by Fumitaka Nagao, K 209 ( TFM: S 17007); OITA Prefecture, Saiki City, under Quercus spp. , 17 Oct 2003, collected by Yoichi Sunada, K 401 ( TFM: S 17008); Yufuin-cho, 29 Sep 2007, collected by Atsuko Hadano and Hiromi Sasaki, K 225 ( TFM: S 17010).

Additional comments: T. longispinosum have more five-spored asci than the other species, but the frequency of five-spored asci varies depending on specimens; observations of two or more specimens are needed. Sakae Takayama and Shoichi Yoshimi first found a black truffle in Japan and identified it as T. indicum (Japanese name “ Iboseiyoshoro ”) [ 42]. The ascospores exhibit surface ornamentation with conspicuously long spines that were 4–8 (–10) μm high with 4 μm bases, which correspond to the T. longispinosum characters. Therefore, we assigned the Japanese “ Iboseiyoushoro ” to T. longispinosum .

Tuber himalayense B.C. Zhang & Minter, Trans. Br. Mycol. Soc. 91(4): 595 (1988).

MycoBank MB 134661. Fig 7A–7E = Tuber formosanum H.T. Hu & Y. Wang, Mycotaxon 123: 296 (2013).

MycoBank MB 563693

Etymology: Japanese name “ Asiakuroseiyoshoro ” from Asia = locality, Kuro = black, seiyoshoro = Japanese name for the genus Tuber ).

Ascomata: hypogeous, 20–60 mm in diam, subglobose and slightly lobed, firm, brown (10 R 4/8) to dark greyish (7.5YR 8/18), with low polygonal warts, 4–6 ridges, up to 500-μm high. Odor: aromatic, similar to seaweed or laver boiled in soy sauce when mature. Peridium: 400– 800 μm thick, variable, pseudoparenchymatous, composed of two layers: outer layer 150–

200 μm thick, composed of irregular or ellipsoidal cells 10–20 × 5–15 μm, with thick, dark brown 1–2-μm walls; inner layer 200–600-μm thick, composed of hyaline to yellowish, polygonal cells 5–15 × 5–10 μm that merge with glebal tissue of interwoven hyphae. Gleba: solid, whitish when young, becoming dark brown to blackish at maturity, marbled with distinct, whitish, meandering veins that merge at many points. Interwoven hyphae of glebal tissue:

3–7 μm broad with scattered cells, gelatinized, inflated up to 10 μm. Asci: typically subglobose to broadly ellipsoid, occasionally ellipsoid, variable depending on number of spores, 48–

81 × 38–73 μm (n = 164), rarely stipitate, 1–4(–5)-spored. Ascospore: mostly ellipsoid, rarely globose, whitish or hyaline when young, becoming light brown (5 Y 8/3) to dark brown (5YR9/ 4) at maturity, 31–41 × 24–35 μm, Q = 1.1–1.6 (1-spored, n = 7); 24–44 × 17–32 μm, Q = 1.0– 1.6 (2-spored, n = 22); 17–37 × 15–33 μm, Q = 1.0–1.8 (3-spored, n = 154); 14–34 × 12–26 μm, 1.0–2.0 (4-spored, n = 279); 18–31 × 14–22 μm, Q = 1.0–1.9 (5-spored, n = 24), excluding ornamentation. Ornamentation with very variable: spines with free, partial reticulate, spiny-reticulate, and alveolate. Spines up to 2–7 (9) μm (n = 508) in height with 3–7-μm bases.

Habitat and distribution: North-western Provinces of India to southern China, Taiwan and Japan. In Japan, the fruiting period is from July to January. Woodland under Betula and Carpinus ( Betulaceae ); Castanea , Castanopsis and Quercus ( Fagaceae ); and Abies and Pinus ( Pinaceae ).

Specimens examined: JAPAN: MIYAGI Prefecture, Sendai City, Dec 2003, collected by Yoko Ando, K 464 ( TFM: S 17011); Sendai City, 23 Oct 2005, collected by Yoko Ando, K 465 ( TFM: S 170012); CHIBA Prefecture, Narashino City, under Q. acutissima , 15 Nov 2015, collected by Hiromi Kinoshita, S 27 ( TFM: S 17013); KYOTO Prefecture, Kyoto City, under Q. glauca and Q. serrata , 6 Dec 2004, collected by Takashi Yamanaka and Keisuke Obase, S 4 ( TFM: S 17014); HYOGO Prefecture, Sanda City, under Q. glauca , 27 Nov 2015, collected by Mitsuo Nabe and Michiyo Nabe, S 17 ( TFM: S 17015); OKAYAMA Prefecture, Niimi City, under Carpinus tschonoskii and Q. serrata , 19 Dec 2015, collected by Hideo Hara, S 23 ( TFM: S 17016); KOCHI Prefecture, Umaji Village, under Q. glauca and Q. serrata , 3 Feb 2017, S 66 ( TFM: S 17017); EHIME Prefecture: Futami-cho, under Castanopsis sieboldii and Quercus sp. , 24 Nov 2006 collected by Fumitaka Nagao, K 152 ( TFM: S 17018); OITA Prefecture, Yufu City, 24 Oct 2008, collected by Atsuko Hadano and Eiji Hadano, K 307 ( TFM: S 17019); Yufu City, under Q. acutissima and Q. serrata , 8 Oct 2006, collected by Hiromi Sasaki, K 448 ( TFM: S 17020).

Additional comments: Hu [ 4] described T. formosanum from Taiwan as a distinct species based on morphological observation; subsequently, Qiao et al. [ 6] typified T. formosanum based on a newly collected sample, because there was no typification in the original description by Hu [ 4]. They denoted that T. formosanum differs from T. indicum by its asci with a short stipitate, spiny-reticulate ascospores and association with Cyclobalanopsis glauca (= Quercus glauca ) [ 6]. However, we showed that T. formosanum is phylogenetically and morphologically indistinguishable from T. himalayense (= T. indicum group B) and Tuber sp. 6 . Because T. himalayense was described by Zhang & Minter [ 7] before T. formosanum was described by Hu [ 4], we synonymize T. formosanum with T. himalayense (hereafter we call Tuber sp. 6 and T. formosanum as “ T. himalayense ”).

Discussion Phylogenetic analyses of the T. indicum complex have been conducted based on ITS, LSU, Pro-

tein Kinase C, β- tublin, mcm7, and TEF-1α sequences [ 8, 9, 13, 14, 15, 43], and all analyses showed two distinct lineages referred to as T. indicum groups A and B. Here, we provide the first MAT phylogenies for the T. indicum complex, including Japanese specimens. Three independent lineages were resolved: T. indicum , T. longispinosum , and T. himalayense ; this was also confirmed in the three-locus phylogeny ( ITS, β- tublin, and TEF 1-α). The T. himalayense clade was composed of specimens that had mainly spine or pseudoreticulum spore ornamentations, and some specimens exhibited a rather complete reticulum, such as the T. himalayense type specimen [ K ( M)33236] [ 7] ( S 1 Fig). Alternatively, the specimens that belonged to the T. indicum clade generally had the same morphological characters as those of the T. himalayense clade, but had no complete reticulum ornamentation. This corresponds to the characters of the T. indicum type specimen [ 7, 14]. Thus, our phylogenetic and morphological analyses revealed that the specimens that belonged to T. indicum and T. himalayense clades were generally consistent with the findings of the previous studies.

Figures

Fig 6. Tuber longispinosum photographs (holotype, TFM: S17009). A. Dried ascomata (bar = 1 cm). B. Fruit bodies photographed in the field.C. Peridial warts, (bar = 3 mm). D. Asci and ascospores (bar = 30 μm). E. Ascospore (bar = 10 μm). F. Peridium in cross section (bar = 50 μm). https://doi.org/10.1371/journal.pone.0193745.g006

Fig 7. Tuber himalayense photographs (TFM: S17015). A. Ascomata (bar = 1 cm). B. Peridial warts, (bar = 3 mm). C. Asci and ascospores (bar = 30 μm). D. Peridium and glebal tissue in cross section (bar = 50 μm). E. Ascospore (bar = 10 μm). https://doi.org/10.1371/journal.pone.0193745.g007