mycoscience 57 (2016) 393e399

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Short communication Tuber shidianense and T. calosporum, two new truffle species from southwest China

* Shan-Ping Wan a,b, Xiang-Hua Wang c, Yi Zheng a, Fu-Qiang Yu b, a College of Resource and Environment, Yunnan Agricultural University, Kunming 650100, PR China b Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China c Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China article info abstract

Article history: Based on morphological and molecular phylogenetic analyses, two new species, Tuber Received 26 June 2015 shidianense and T. calosporum, are described from southwest China. They are clearly Received in revised form different from any other taxon in the genus Tuber. Tuber shidianense belongs to the 8 December 2015 Puberulum group. It is characterized by whitish ascomata, pseudoparenchymatous Accepted 22 June 2016 peridium and globose ascospores with deep alveolar ornamentation. Tuber calosporum be- Available online 11 August 2016 longs to the Macrosporum group. It is recognized by yellow and brown ascomata, pros- enchymatous peridium and large elliptic ascospores with low alveolate wall. Phylogenetic Keywords: relationships and morphological differences between these two new species and their Macrosporum group related species are discussed. Phylogenetic analysis © 2016 Published by Elsevier B.V. on behalf of The Mycological Society of Japan. Puberulum group Taxonomy

Tuber F.H. Wigg. (Ascomycota, Pezizales) is one of the most studies (Garcı´a-Montero et al. 2010). Recently, a large number important hypogeous fungi in terms of both economic and of new Tuber species, a large proportion of which are white ecological value. Some Tuber species, such as T. magnatum truffles, have been discovered and described by Chinese my- Pico, T. melanosporum Vittad., T. aestivum Vittad. and T. indicum cologists from southwest China (Fan et al. 2011, 2012a,b,c,d, Cooke & Massee, have high culinary value because of their 2013, 2014, 2016a,b; Fan and Cao 2012; Deng et al 2013; Fan unique scent. Tuber species form symbiotic associations with and Yue 2013; Li et al 2014). Morphological observations, in angiosperms and gymnosperms and play an important role in some cases with sequence analyses of the nuclear ribosomal ecosystems (Pomerico et al 2006; Wang and Liu 2009; Trappe internal transcribed spacer (ITS), showed that these species and Claridge 2010). In China, new species of the genus Tuber mainly belong to the Puberulum group. The others include were first reported in the 1980s (Liu 1985). In 2010, Garcı´a- members of the Latisporum, Rufum, Maculatum, Macro- Montero characterized 16 Chinese Tuber taxa by locating and sporum and Melanosporum groups. In this study, we describe analyzing the available scientific information from previous two new Tuber species belonging to the Puberulum and

* Corresponding author. Tel./fax: þ86 0871 65223085. E-mail address: [email protected] (F.-Q. Yu). http://dx.doi.org/10.1016/j.myc.2016.06.007 1340-3540/© 2016 Published by Elsevier B.V. on behalf of The Mycological Society of Japan. 394 mycoscience 57 (2016) 393e399

Macrosporum groups. Morphological comparisons with Criterion (AIC) (Akaike 1974) implemented by MrModeltest closely related taxa are presented, and their molecular v.2.3 (Nylander 2004). Bayesian analysis was carried out using phylogenetic position in Tuber is examined. the selected model with four chains sampled every 100 gen- Fresh samples were collected from forests of Pinus yunna- erations and run for a total of 2,000,000 generations. The nensis Franch. and a local mushroom market in Yunnan, average standard deviations of split frequencies were less China. Morphological descriptions were based on fresh ma- than 0.01 at the end of the run and ESS (effective sampling terials and microscopic examination on dry material size) values were >200. A majority rule consensus tree was following the methods of Yang and Zhang (2003). Sections built after discarding trees from a 25% burnin. Posterior were made with a razor blade by hand, mounted in a 5% (w/v) probabilities (PPs) were calculated using the sumt command KOH solution and examined under a light microscope (Leica implemented in MrBayes. DM2500, Leica Microsystems, Shanghai, China). For scanning The aligned ITS dataset includes 80 samples and 663 electron microscopy (SEM), spores were scraped from the characters: 257 of ITS1 (complete), 155 of 5.8S (complete) and dried gleba onto doubled-sided tape, which was mounted 251 of ITS2 (complete). The ML analysis resulted in a best tree directly on an SEM stub, coated with gold-palladium, exam- with a likelihood of 3346.27. Bayesian analysis ran for a total ined and photographed with a JEOL, JMS-5600LV SEM (JEOL, of 2,000,000 generations to get convergence. ML and Bayesian Shanghai, China). For evaluation of the range of spore-size, 40 analyses yielded similar tree topologies, and only the tree ascospores each from one specimen of each collection cited inferred from the ML analysis is shown. The result revealed were measured. In the description of the ascospores, the six distinct groups, which covered most of the Chinese col- abbreviation Q represents the range of ratio of spore length to lections (Fig. 1). Phylogenetically, T. shidianense belongs to the spore width calculated for each spore and Qm is the average of Puberulum group and is sister to the subclade containing T. the Q values. The specimens have been deposited at the jinshajiangense L. Fan and T. sinosphaerosporum L. Fan, J.Z. Cao & Herbarium of Cryptogams, Kunming Institute of Botany, Chi- Yu Li (subclade A). This subclade is supported by a BS value of nese Academy of Sciences (KUNeHKAS). 72% and a PP of 0.98. Tuber shidianense is closely related to T. Total DNA was extracted from pieces of dried ascomata jinshajiangense with high BS (90%) and PP (1.0) values. DNA with a modified CTAB procedure (Gardes and Bruns 1993). sequence analysis showed that these three species share a Polymerase chain reactions (PCR) were performed using the 86.9%e88.9% similarity in their ITS sequences. Tuber calo- primer combination ITS1F/ITS4 (White et al 1990; Gardes and sporum belongs to the Macrosporum group and is grouped Bruns 1993). In 25 mL of PCR reaction solution contained 1 mL with T. glabrum L. Fan & S. Feng and T. sinomonosporum J.Z. Cao þ DNA, 1 mL(5mM) of each primers, 2.5 mL10 buffer (Mg2 plus), & L. Fan with strong support values (BS ¼ 100%, PP ¼ 1.0). ITS m m m 1 L dNTP (1 mM), 0.5 L BSA (0.1%), 0.5 L MgCl2,1UofTaq sequence analysis showed that these three species share a DNA polymerase (Takara Tag, Takara Biotechnology, Dalian, 87.2%e89.9% similarity. China). PCR reactions were run as follows: 94 C for 5 min, followed by 35 cycles of 94 C for 30 s, 52 C for 1 min and 72 C Tuber shidianense S.P. Wan, sp. nov. Fig. 2. for 1 min. The final reaction was followed by an extension at MycoBank no.: MB814191. 72 C for 10 min. The PCR products were sent to Sangon Biotech Corporation (Shanghai, China) for purifying and Ascomata whitish and brown, cracked; Dermatocystidia sequencing by using ITS1F/ITS4. dense; Ascospores globose with deep alveolar ornamentation. A total of 78 Tuber ITS rDNA sequences were used for Type: CHINA, Yunnan Province, Shidian County (11. 99E, phylogenetic analysis (Supplementary Table 1), including 2 30. 24N), in humous soil under mixed forest with Pinus yun- sequences of T. shidianense, 4 sequences of T. calosporum and nanensis as dominant species, at about 2335 m, 3 Nov 2014, 72 sequences were downloaded from GenBank. Two se- (holotype, wsp256, HKAS88770). quences derived from Choiromyces meandriformis Vittad. Gene sequences ex holotype: KT444595 (ITS); ex paratype: (HM485330 and HM485331) were selected and used as out- KT444596 (ITS). groups (Fig. 1). The matrix has been deposited in TreeBASE Etymology: In reference to the location of the type under the accession number S19191. collection. Sequences were edited and assembled using SeqMan II Ascomata (Fig. 2A) 0.5e2 cm in diam, subglobose or oval- (Larsson and Sundberg 2011). Alignment was performed using shaped, firm and occasionally cracked. Peridium densely pu- the online version of the multiple sequence alignment pro- bescent, whitish, ecru to pale yellowish-brown when fresh, gram MAFFT v7 (Katoh and Toh 2008), applying the L-INS-I becoming brown when dried. Gleba solid, pale gray when strategy, and the alignments were manually adjusted in Bio- young, marbled with white veins. Odor pleasant. Taste not Edit. The phylogenetic relationships of taxa were inferred recorded. using maximum likelihood (ML) and Bayesian inference (BI). Peridium 180e340 mm thick, two layers, the outer layer ML analysis on the ITS dataset was performed in RAxML 100e270 mm thick, pseudoparenchymatous (Fig. 2C, E), v.7.2.6 (Stamatakis 2006) and the GTR þ GAMMA substitution composed of big, subglobose to subangular cells, model with parameters unlinked. ML bootstrap (BS) replicates 6.8e37 8e29 mm, pale yellow or hyaline; the inner layer (1000) were computed in RAxML with a rapid bootstrap anal- 39e260 mm thick, composed of intricately interwoven, hyaline ysis and search for the best-scoring ML tree. Bayesian analysis and thin-walled hyphae, 1.5e5 mm in diam. Dermatocystidia, on the ITS dataset was performed in MrBayes v.3.1.2 (Ronquist radial, up to 75 mm long, 7 mm in diam, dense, multi-septate, and Huelsenbeck 2003) and the GTR þ I þ G model was tapered, hyaline to whitish (Fig. 2E). Gleba composed of hya- selected as the best model under the Akaike Information line, interwoven, thin-walled hyphae, 1.5e5 mm broad at the mycoscience 57 (2016) 393e399 395

100/1.000/1.0 T.T. bbrunneumrunneum KKT897478T897478 83/- T.T. bbrunneumrunneum KKT897479T897479 T.T. ppseudoseparansseudoseparans KKT89T8974807480 100/1.0 87/-87/- T.T. ppseudoseparansseudoseparans KKT897481 98/1.0 T.T. bbonitoionitoi KKT897473T897473 100/1.000/1.0 T.T. bbonitoionitoi KT897472KT897472 95/0.99 T.T. ttequilanumequilanum KT897487KT89747 87 T.T. ttequilanumequilanum KT897486KT8974 100/1.0 94/1.04/1.0 T.T. a anniaenniae HHM485338M485338 T.T. aanniaenniae HHM48533M485339 -/0.98 100/1.01 /1.0 T.T. hhuizeanumuizeanum JN870100JN870100 T.T hhuizeanumuizeanum JQ910651JQ91 100100 T.T. lliuiiui DDQ898182Q898182 1.0 T.T. lliuiiui DDQ478636Q478636 100100 T.T. lliyuanumiyuau num JJQ771193Q771193 1.0 T.T. zzhongdianensehongdgdianense DDQ898186Q898186 100/1.000/1.0 T.T. zzhongdianensehongddianense NNR119621R1196 100/1.0100/1.0 T.T. ddryophilumryophilum JJQ925644Q925644 80/0.96 T.T. ddryophilumryophilum HHM485353M485353 97/- T.T. ppuberulumuberulum FFM205695M205695 Puberulum 100/1.0 T.T. ppuberulumuberulum FFM205643M20 T.T. bborchiiorchii AAJ557540J557540 Group T.T. bborchiiorchii AAJ557541J557541 100/1.0 94/1.0 T.T. ssphaerosporuphaerosporum HHM485390M485390 T.T. ssphaerosporumphaerosporum GGQ221449Q221449 T.T. ccalifornicumalifornicum HHM485350M485350 100/1.00/1.0 T.T ccalifornicumalifornicum HHM485346M485346 100/1.0 T.T. ooligospermumligospermum FFM205509M205509 T.T. ooligospermumligog spermum FFM205508M20550 90/1.0 100/1.0 T.T. sshidianensehidianense KKT444596T444596 -/0.98 T.T. sshidianensehidianense KKT444595T444595 ( (holotype)ho T. jinshajiangense KP276177 A 100100 72/0.98 T.T. ssinosphaerosporuminnosphaerosporum JJX092087X092087 1.0 T.T. ssinosphaerosporuminosphaerosporum JJX092086X0920 94/1.094/1 0 T.T. vvesicoperidiumesicoperiddium JQJQ690071690071 79/- T.T. vvesicoperidiumesicoperiddium JQ690072JQ690072 100100 T.T. mmicrosphaerosporumicrosphaerosporum KKF805726F8 1.0 9898 T.T. ssinopuberuluminopuberulum JJQ690073Q6900 -/0.96 1.0 T.T. llijiangenseijiangense KKF805727F805727 T.T. xxuanhuaenseuanhuaense KPKP276179276179 100/1.0100/1. T.T. lalatisporumtisporum DQ898185DQ898185 100/1.0 T.T. llatisporumatissporum DDQ898184Q898184 T.T. ppanzhihuanenseanzhihuanense JQJQ978654978654 100/1.0 T. panzhihuanenseanzhihuanense JQJQ978655978655 T.T. ppseudosphaerosporumseudosphaerosporum KKF744063F7 Latisporum T.T. aalboumbilicumlboumbilicum KKJ742702J742702 Group 98/1.0 9191 100/1.0 T.T. pparvomurphiumarvomurphium KPKP276186276186 1.0 TT.. pparvomurphiumarvomurphp ium KPKP2761852 100/1.0 T.T. ccaoiaoi KKP276183P276183 T.T. ccaoiaoi KKP276181P276181 100/1.0100/1 T.T. mmaculatumaculatum AAJ557517J557517 89/0.9989/0.99 T. mmaculatumaculatum AAJ557516J557516 95/1.0 T.T. rrapaeodorumapaeodorum AAJ557522J5575 T.T. hhubeienseubeiense KKT067688T067 Maculatum 100/1.0 T.T. ffoetidumoetidum AAJ557544J557544 Group 74/0.99 85/- T.T. mmicroverrucosumicroverrucosum JNJN870099870099 T.T. ppseudomagnatumseudomagnatum KKP276184P276184 100/1.0 T. pseudomagnatumagnatum KKT067686T067686 T.T. ccalosporumalosporum KKT444599T444599 T.T. ccalosporumalosporum KKT444600T444600 100/1.0 T.T. ccalosporumalosporum KKT444597T444597 T.T. ccalosporumalosporum KKT444598T444598 (holotype)(holot B 100/1.0 100/1.0 T.T. gglabrumlabrum KKF002727F002727 Macrosporum T.T. gglabrumlabrum KF002731KF002731 100/1.0 100/1.0 T.T. ssinomonosporuminomonospporum KKF002729 Group 100/1.0 T.T. ccanaliculatumanaliculatum GGQ221456Q221456 T.T. ccanaliculatumanaliculatum GGQ221455Q221455 100/1.0 T.T. mmacrosporumacrosporum KKJ524532J524532 T.T. mmacrosporumacrosporum KKJ524529J524529 98/1.098 T.T. lliaotongenseiaotongense GGU979037U979037 83/0.983/0 T.T. hhuidongenseuidongense FFJ797877 Rufum 100/1.0 100100 T.T. rrufumufum EEF362477F36247 Group 1.0 T.T. rrufumufum EEF362474F362474 98/1.0 T.T. ttaiyuanenseai uanense GGU979033U979033 100/1.01000 T.T. iindicumndicum JJQ639005Q639005 99/1.0 T.T. iindicumndicum JJQ639007Q639007 Melanosporum 100/1.0 T. melanosporumosporum KKM659873M659873 Group T. pseudoexcavatum DQ329374 C.C. m meandriformiseandriformis HHM485331M4853 C. meandriformis HM485330 0.1

Fig. 1 e RAxML tree based on ITS sequences of 78 taxa of Tuber. Bootstrap (BS) values derived from Maximum Likelihood (ML) analysis (≥70%) and Posterior Probabilities (PPs) from Bayesian Inference (≥0.90) are shown above or beneath the branches at nodes. Tuber shidianense and T. calosporum are indicated in boldface. Species are labeled by their Latin binomial. septa, the cells cylindrical interwoven to inflated, 2e5 mm 41e43 40e42.5 mm, in 2-spored asci 36e40(e41) (35.5e) broad. Asci (Fig. 2B, D) (64e)68e100 52e87 mm, globose to 36e40 mm, in 3-spored asci 26e36(e36.5) (25.5e)26e36 mm, subglobose, pyriform, ellipsoid or irregular, hyaline, sessile or and in 4-spored asci (23e)24e28.5 22e26(e28) mm; e m e ¼ e ¼ ± with a short stalk, walls thin (1 2 m thick), 1 4 spored. As- Q 1 1.09, Qm 1.01 0.03; ornamented with regular alve- cospores (Fig. 2B, D, F), globose, rarely subglobose, hyaline olate reticulum, 3e7.7 mm deep, constituted of pentagonal when young, becoming yellowish brown at maturity; in 1- meshes 8.5e15(e16) 5.2e12 mm, 3e5 meshes across the spored asci excluding alveolar ornamentation spore width. 396 mycoscience 57 (2016) 393e399

Fig. 2 e Tuber shidianense (HKAS88770, holotype). A: An ascoma and its gleba. B, D, F: Asci and ascospores. C, E: Peridium sections. E: Dermatocystidia. F: A SEM photo of an ascospore. Bars:A1cm;BeF20mm.

Further specimens examined, CHINA, Yunnan Province, Shi- becoming brown when dried. Gleba solid, brown when dian County, 3 Nov 2014, S.P. Wan, wsp352 (HKAS88771). mature, marbled with white veins. Odor pleasant. Taste not recorded. Tuber calosporum S.P. Wan, sp. nov. Peridium (Fig. 3B) 123e164 mm thick, prosenchymatous, MycoBank no.: MB814192. Fig. 3. composed of subglobose cells and interwoven hyphae, cells yellowish, 1e4 layers, (5.6e)6e34 2.2e13(e14) mm, hyphae Ascomata brownish-yellow, verrucose with superficial 1.2e5.5 mm in diam. Gleba composed of hyaline, interwoven, yellow furrows; Ascospores large, ellipsoid with shallow thin-walled hyphae, 2.5e6 mm broad at the septa, the cells alveolar ornamentation. cylindrical interwoven to inflated, 2e6 mm broad. Asci (Fig. 3C, Type: CHINA, Yunnan province, Huize County, in soil E) 102e149(e151) (82e)84e147 mm, ellipsoid, rarely pyriform under mixed forest with Pinus yunnanensis as dominant spe- or irregular, hyaline, mostly 1-spored, occasionally 2- or 3- cies, Oct 2014 (provided by the sellers), (holotype, wsp143, spored. Ascospores (Fig. 3CeE) ellipsoid, at first hyaline or HKAS88790). yellowish, becoming reddish-brown at maturity; in 1-spored Gene sequences ex holotype: KT444598 (ITS); ex paratype: asci excluding alveolar ornamentation 77e101(e102) KT444597 (ITS), KT444599 (ITS), KT444600 (ITS). 57e72(e74) mm, in 2-spored asci 60e74(e76) 45e54(e56) mm, Etymology: In reference to the beautiful ornamentation of and in 3-spored asci (57e)59e61(e62) (37e)39e46 mm; ¼ e ¼ ± the ascospores. Q 1.31 1.54, Qm 1.38 0.04; ornamented with an shallow Ascomata (Fig. 3A) 1.4e2.3 cm in diam, globose, sub- alveolate reticulum, less than 2 mm deep, constituted of globose or oval-shaped, firm. Peridium verrucose, yellow and irregularly pentagonal or hexagonal meshes, 4e9 across the brown, with yellow furrows of crumbs when fresh, ascospore width. mycoscience 57 (2016) 393e399 397

Fig. 3 e Tuber calosporum (HKAS88790, holotype). A: An ascoma and its gleba. B: A peridium section. C, E: Asci and ascospores. D: A SEM photo of an ascospore. Bars: A 1 cm; B, D, E 20 mm; C 50 mm.

Further specimens examined: CHINA, Yunnan province, prosenchymatous peridium while T. shidianense has the Huize County, Oct 2014, S.P. Wan, wsp145 (HKAS88751), pseudoparenchymatous type (Moreno-Arroyo et al. 2000). wsp186 (HKAS88791), wsp382 (HKAS88794). Tuber sphaerosporum Gilkey differs in having a glabrous peridium while that of T. shidianense is densely pubescent Morphologically, T. shidianense shares characteristics with (Gilkey 1954). Tuber bonitoi Guevara & Trappe and T. brunneum some species of the Puberulum group: whitish, hairy asco- Guevara, Bonito & Trappe sometimes have globose spores, but mata, pseudoparenchymatous peridium and reticulate mainly have broadly ellipsoid spores (Guevara-Guerrero et al. spores. Nevertheless, T. shidianense has globose (occasionally 2015). Tuber californicum Harkn. and European T. puberulum ¼ e ¼ ± subglobose) spores (Q 1 1.09, Qm 1.01 0.03), which can Berk. & Broome occasionally have broadly elliptic spores distinguish it from other species with broadly elliptic spores (Gilkey 1954; Bidaud and Vooren 2008). Asian T. xuanhuaense L. (T. borchii Vittad., T. dryophilum Tul. & C. Tul., T. huizeanum L. Fan collected from central China (Hubei province) differs in its Fan & Yu Li, T. anniae W. Colgan & Trappe, T. zhongdianense prosenchymatous peridium (Fan et al. 2016a). The T. lijiangense X.Y. He, Hai M. Li & Y. Wang) (Mello et al. 2000; He et al. 2004; L. Fan & J.Z. Cao complex (T. microsphaerosporum L. Fan & Y. Li, Chen and Liu 2007; Fan et al. 2012d; Wang et al. 2013), elliptic T. lijiangense, T. vesicoperidium L. Fan, T. sinopuberulum L. Fan & spores (T. liyuanum L. Fan & J.Z. Cao, T. liui A S. Xu) (Xu 1999; J.Z) (Fan et al. 2016a) were described from southwestern Fan and Cao 2012) and fusiform spores (T. pseudoseparans China, all these species have globose spores, but they can be Guevara, Bonito & Trappe, T. tequilanum Guevara, Bonito & distinguished from T. shidianense by other morphological fea- Trappe) (Guevara-Guerrero et al. 2015). tures. Tuber microsphaerosporum has more spores (1e7) in its Although globose or subglobose spores are also common in asci, lower alveolate wall (2.5e3 mm) and more meshes (3e8) the Puberulum group, differences can be found to differentiate across the spore width (Fan et al. 2012b) while T. shidianense these species from T. shidianense: North American T. oligo- has 1e4 spores in its asci, 3e7.7-mm-deep meshes and 3e5 spermum (Tul. & C. Tul.) Trappe differs in its one-layer meshes across the spore width. Tuber lijiangense is very similar 398 mycoscience 57 (2016) 393e399

to T. shidianense in ascocarps, peridium and spore shape, but calosporum extremely closely with its red-brown, large, ellip- differs in its lower alveolate wall, which are usually less than tical and low-meshed ascospores, but can be easily distin- 2 mm deep (Fan et al. 2011). Tuber vesicoperidium differs in its guished by its smooth ascomata with whitish furrows in prosenchymatous peridium. Tuber sinopuberulum has glabrous contrast to the verrucose ascomata of T. calosporum with su- peridium and sometimes has ellipsoid ascospores (Fan et al. perficial yellow furrows. Phylogenetically, although T. glabrum 2012c). The differences between T. shidianense and the spe- is closely related to T. calosporum, this relationship is not cies described above are not only based on morphological supported by a high BS value (Fig. 1 subclade B). Tuber sino- analyses, but also on the strength of molecular evidence monosporum forms a single branch, which is separated from T. (Fig. 1). The ITS-based ML tree shows that each of these spe- calosporum and T. glabrum with high BS (100%) and PP (1.0) cies is phylogenetically distant from T. shidianense. values (Fig. 1 subclade B). Morphologically, T. sinomonosporum Phylogenetically, T. shidianense forms a distinct branch differs in having globose spores compared with T. calosporum with 100% BS support and 1.0 PP and clusters with Chinese T. and T. glabrum. Moreover, the similarities in ITS sequences jinshajiangense and T. sinosphaerosporum in subclade A (Fig. 1). among these three Chinese species are less than 90%. This Tuber jinshajiangense described from southern China also has indicates that T. calosporum represents a different taxon from globose spores (Fan et al. 2012a), and is closely related to T. the known species of the Macrosporum group. It is worth shidianense with BS of 90% and PP of 1.0. However, in mentioning that T. gigantosporum Y. Wang & Z.P. Li is another morphology T. jinshajiangense has smooth peridium, relatively species that is similar to T. calosporum in having brown asco- smaller spores (22.5e37.5 mm), lower alveolate wall (2.5e5 mm) mata and large reddish-brown, shallow reticulate, elliptical and more meshes (4e6) compared with T. shidianense. ascospores. However, T. gigantosporum is clearly separated Although T. sinosphaerosporum is sister to the subclade A from the new species by its larger spores, up to 120 mm in size containing T. shidianense and T. jinshajiangense, this relation- (Wang and Li 1991). ship is not supported by a high BS value (Fig. 1). In addition, T. Given the variability and overlap of the morphological sinosphaerosporum is morphologically distinct from T. shidia- characters among the species in Tuber, sometimes classical nense, having larger meshes (2e4 across the spore width), delimitation of the taxa is difficult because similar related glabrous peridium and convolute ascomata with several deep species cannot easily be distinguished. However, detailed furrows (Fan et al. 2012a). Furthermore, DNA analysis revealed comparisons of macroscopic or microscopic characteristics less than 90% ITS sequence similarity among T. shidianense, T. show that T. shidianense and T. calosporum are well separable jinshajiangense and T. sinosphaerosporum. Thus, T. shidianense is from all other known taxa of Tuber, and we have tested the well separated from the known species of the Puberulum validity of these identifications using molecular evidence, group and represents a new taxon on the basis of both which implies that T. shidianense and T. calosporum represent morphological and molecular evidence. novel species. In addition to the above description, T. pseudosphaer- osporum L. Fan, T. parvomurphium L. Fan and T. caoi L. Fan, which belong to the Latisporum group (Fig. 1), also have Acknowledgments globose spores (Fan and Yue 2013; Fan et al. 2016a). According to Fan et al. (2016a), the Latisporum group includes species The work was supported by National Natural Science Foun- known from eastern Asia only, and probably represents a new dation of China (Nos. 31370070 and 31460551) and the Joint lineage that arose in parallel with the Puberulum, Maculatum Funds of the National Science Foundation of China and and Gibbosum lineages. Through morphological analysis and Yunnan Province Government (No. U1202262). comparison, T. pseudosphaerosporum and T. parvomurphium are different from T. shidianense by their glabrous peridium, T. caoi Appendix A. Supplementary data by its green gray ascomata, smaller spores (25e40 mm) and more meshes (4e7). Moreover, T. maculatum Vittad., which Supplementary data related to this article can be found at was transferred to the Maculatum group from the Puberulum http://dx.doi.org/10.1016/j.myc.2016.06.007. group according to Bonito et al. (2013), is difficult to distin- guish from T. shidianense when they are fresh, but T. mac- ulatum is distinct in having a glabrous peridium, ellipsoid references ascospores and a one-layer prosenchymatous peridium structure. Tuber calosporum is well distinguished from species of other Akaike H, 1974. A new look at the statistical model identification. groups by its verrucose ascomata, mostly 1-spored asci with IEEE Transactions on Automatic Control 19: 716e723; http:// large, shallowly reticulate and elliptical ascospores. According dx.doi.org/10.1109/TAC.1974.1100705. to our phylogenetic analysis, four samples of T. calosporum Bidaud A, Vooren NV, 2008. Tuber puberulum,recolt eenIs ere. unite and cluster with Chinese T. glabrum and T. sinomono- Bulletin mycologique et botanique Dauphine-Savoie 189: 45e48. sporum in the Macrosporum group, and these together form a Bonito G, Smith ME, Nowak M, Healy RA, Guevara G, Cazares E, single subclade with 100% BS support and PP of 1.0 (Fig. 1 Kinoshita A, Nouhra ER, Dominguez LS, Tedersoo L, Murat C, Wang Y, Moreno BA, Pfister DH, Nara K, Zambonelli A, subclade B). These Chinese species group together with the Trappe JM, Vilgalys R, 2013. 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