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Lin et al. Bot Stud (2018) 59:25 https://doi.org/10.1186/s40529-018-0241-y ORIGINAL ARTICLE Open Access Tuber elevatireticulatum sp. nov., a new species of whitish trufe from Taiwan Chieh‑Lung Lin1, Ming‑Jer Tsai2,3, Chuen‑Hsu Fu4, Tun‑Tschu Chang4, Hoi‑Tung Li5 and King‑Fai Wong6* 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 trufe belongs to Puberulum clade was collected from roots of Keteleeria fortunei var. cyclolepis in central Taiwan and appeared to difer from the two recorded species. Results: The whitish trufe is herein described as a new species Tuber elevatireticulatum, which is distinguished from closely resembled Asian whitish trufes species like Tuber thailandicum, T. panzhihuanense, T. latisporum and T. sinopu- berulum 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 signifcant incongruence. Conclusions: The whitish trufe is herein described as a new species T. elevatireticulatum based on the evidence from morphology and DNA sequences. T. elevatireticulatum is the frst scientifc record of whitish trufe in Taiwan. Keywords: Keteleeria, Morphology, Phylogeny, Taxonomy, Taiwan, Trufe, Tuber Background them among the most famous and demanding trufes in True trufes, belonging to the genus Tuber (Tuberaceae, the world (Hall et al. 2007; Bonito et al. 2010a). Pezizales, Pezizomycetes), produce hypogeous asco- Index Fungorum (http://www.index​fungo​rum.org/ carps, which are formed in soil or sometimes within names​/Names​.asp) lists out three hundred and fve layers of leaf litter. Tey have lost the ability to actively Tuber names, however, many of them required clarif- discharge ascospores (Bonito and Smith 2016). Tey are cation (Suwannarach et al. 2015; Kinoshita et al. 2016). symbiotic fungi that develop association with fne roots Bonito et al. (2013) reassessed the published names and of specifc host trees (T. oregonense Trappe, Bonito and estimated 180–220 accepted species in the genus, was P. Rawl. with Douglas fr) or broad host ranges (T. aes- subdivided into 11 major clades according to their phy- tivum (Wulfen:Fr.) Spreng. with some plant species in logenetic relationships. Puberulum clade, Maculatum Betulaceae, Corylaceae, Fagaceae, Tiliaceae, Pinaceae and clade and closely related lineage Gibbosum clade were Cistaceae) (Hall et al. 2007). Te unique aroma makes phylogenetically grouped with as Puberulum Group some species greatly sought after as high-end culinary and members of this group commonly called “whit- ingredients throughout the world, especially in Europe ish trufe” in order to distinguish them from Italian (Hall et al. 2007). Te scarcity and irreplaceably scent of white trufe (T. magnatum in Aestivum clade) (Bonito French Périgord black trufe (T. melanosporum Vittad.) et al. 2010a; Lancellotti et al. 2016). Researches in Tuber and Italian Alba white trufe (T. magnatum Pico.) render 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 *Correspondence: [email protected] are poorly documented, with only T. formosanum Hu 6 Advance Plant Protection Limited Company, Hsinchu, Taiwan Full list of author information is available at the end of the article (invalidly described in 1992 due to the lack of designated © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Lin et al. Bot Stud (2018) 59:25 Page 2 of 10 holotype and later re-typifcation in 2013) and T. furfu- Molecular analysis raceum Hu and Wang reported previously. Both species DNA extraction form symbiotic association with roots of Quercus glauca Approximately 9–14 mg of gleba tissue of fresh ascocarps (Tunb. ex Murray) Oerst. in the family of Fagaceae (Hu were ground by plastic pestle with 800 µl of Lysis Bufer 1992; Hu and Wang 2005; Qiao et al. 2013). A whitish (Taiwan Advanced Nanotech Inc.; containing Guani- trufe was mentioned in Hu (1987) but lacks a formal dine salt, Tris bufer and surfactants) in 1.5 ml centrifuge description. tube for DNA extraction. DNA was then extracted using During our survey of hypogenous fungi in Taiwan, a the TANBeadⓇ fungal Nucleic Acid Extraction Kit and whitish trufe was found under Keteleeria fortunei var. TANBeadⓇ Nucleic Acid Extractor (Taiwan Advanced cyclolepis (Flous) Silba, in Sitou Tract, Nantou County of Nanotech Inc.) following protocol of the manufacturer. central Taiwan. It resembles several known Asian whit- ish trufes in the Puberulum Clade, such as T. thailandi- Polymerase chain reaction (PCR) amplifcation cum Suwannarach et al. (2015), T. panzhihuanense Deng and sequencing et al. (2013), T. latisporum Chen and Liu (2007), T. pseu- Two nuclear ribosomal DNA loci were used for amplify- dosphaerosporum Fan and Yue (2013), and T. alboumbili- ing and sequencing, including the internal transcribed cum Wang and Li (Li et al. 2014), but difers from species spacer (ITS) with forward primer ITS5 (5′-GGA AGT in the Puberulum clade by the only species associated AAA AGT CGT AAC AAGG-3′) was paired with reverse with Keteleeria host, small light brown ascocarps with primer ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) hyphae-like hairs arised, dark brownish and elliptical (White et al. 1990); and ribosomal large subunit (LSU) ascospores ornamented with a prominently raised alveo- with forward primer LR0R (5′-ACC CGC TGA ACT late reticulum. TAAGC-3′) (Rehner and Samuels 1994) was paired with reverse primer LR5 (5′-TCC TGA GGG AAA CTTCG-3′) Methods (Vilgalys and Hester 1990). PCR was performed in 25 µl Sample collection reactions containing 2.5 µl DNA template, 1 µl primer Ascocarps were collected with three-pronged garden each, 8 µl ddH20 and 12.5 µl 2× Taq Master Mix (includ- cultivators, wrapped with tissue paper and kept in sep- ing 20 mM KCl, 4 mM MgSO 4·7H2O, 40 mM Tris–HCl arate plastic zipper bags until further morphological with pH 8.8, 0.2% Triton X-100, 20 mM (NH4)2SO4, and molecular analyses in laboratory. Ascocarps were 0.2 mg/ml BSA, 0.4 mM dNTP mix, 100 U/ml Taq DNA weighted freshly within 24 h, and the pH value of adja- Polymerase and stabilizers) (Genomics Bioscience and cent soil were measured by JENCO 6010M pH meter fol- Technology CO., Ltd.). PCR for ITS/LSU were run as lowing protocol of the manufacturer. 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 Morphological analysis at 72 °C for 30 s/1 min by 30 cycles and a fnal extension Ascocarps were cleaned with dry toothbrush, and then at 72 °C for 5/10 min on a multigene thermal cycler (Lab- cut into halves for observing gleba color or color change net International, Inc.). PCR products were checked on under air exposure. Sections of fresh tissue were made agarose gel containing 1.4% agarose and 0.5× Tris–ace- with a razor blade by hand, then mounted in 0.1% (w/v) tate-EDTA (TAE) and stained with 5 µl/100 ml Health- cotton blue in lacto-phenol for describing morpho- view™ nucleic acid stain under UV light by multilmage™ logical characteristics by a Leica DMLB light micro- light cabinet (Alphalmager 2200). Te PCR products scope. Ascospore dimensions, with the ornamentation were sent to Seeing Bioscience Co., Ltd. for purifcation excluded, were based on at least 100 randomly selected and sequencing by Sanger Sequencing Method (ABI ascospores. Te range of ascospore length to width ratio 3730). (Q), average Q with ± standard deviation (Q) was calcu- lated, and number of meshes across the ascospore width Phylogenetic analyses was measured. Six ITS and eight LSU sequences were obtained For scanning electron microscopy (SEM), ascospores from ascocarps of T. elevatireticulatum and were from dried gleba were mounted onto SEM stubs with submitted to GenBank with Accession Numbers carbon double-sided tape (Nisshin EM CO., Ltd, Tokyo), MF540616–MF540621 (ITS) and LSU sequences: coated with gold–palladium, then examined and photo- LC425119–LC425126 (LSU). Other whitish Tuber graphed with a tabletop HITACHI TM3000 SEM. Holo- sequences were obtained from GenBank database for type was deposited at Herbarium of Taiwan Forestry phylogenetic analyses (Table 1), with Choiromyces alve- Research Institute, Taipei, Taiwan (Index Herbarium: olatus as the outgroup. Sequences were aligned using TAIF). Lin et al. Bot Stud (2018) 59:25 Page 3 of 10 Table 1 Details of the whitish Tuber ITS sequences used in phylogenetic study Taxa Voucher no. Origin GenBank Accession no. References ITS LSU Choiromyces alveolatus MES97 USA HM485332 Bonito et al. (2010a) Choiromyces alveolatus HS2886 USA HM485333 Bonito et al. (2010a) Choiromyces alveolatus p688L USA EU669426 Unpublished Choiromyces alveolatus MES97 USA JQ925660 Bonito et al. (2013) T. alboumbilicum YAAS L2324a China KJ742702 Li et al. (2014) T. bellisporum JT7270 USA FJ809856 FJ809827 Bonito et al. (2010b) T. bellisporum JT6060 USA FJ809857 FJ809828 Bonito et al. (2010b) T. borchii GB45 Italy HM485344 Bonito et al.

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