雲南常見貿易黑塊菌的親緣及分群黃勁暉

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雲南常見貿易黑塊菌的親緣及分群

黃勁暉¹⁾ 傅春旭²⁾ 林介龍³⁾ 張詠怡⁴⁾ 李鎧彤^5,6)

摘要

以產自雲南省之黑塊菌為研究標的，將每顆塊菌子實體作形態鑑定及ITS親緣分析，結果顯示雲南黑塊菌可分為3個分群，分別為擬外孔塊菌分群(Pseudoexcavatum cluster)、印度塊菌分群(Indicum cluster)及臺灣塊菌分群(Formosanum cluster)。2017年的294條黑塊菌ITS序列當中，有76%序列屬於印度塊菌分群，23%序列屬於臺灣塊菌分群，1%序列屬於擬外孔塊菌分群；2016年10條黑塊菌序列當中，80%序列屬於印度塊菌分群，20%條序列屬於臺灣塊菌分群。當中擬外孔塊菌分群可以根據形態及顯微特徵之明顯差異而分辨出來，可是成體很少。另外印度塊菌及臺灣塊菌兩分群，比例接近4:1，

成對比較分析亦得出兩分群間ITS序列相似度有一定的差異(90.56~93.78%)，但比較兩基因分群之子實體，並沒有明顯外觀或顯微特徵可作分辨。高黎貢山產區之黑塊菌表皮包覆的土壤有特別的金屬光澤，亦帶有干貝般的特殊香氣，主要以臺灣塊菌分群的塊菌佔大多數(75%)。

關鍵詞：黑松露、ITS親緣分析、基因分群、成對比較分析、形態鑑定。

黃勁暉、傅春旭、林介龍、張詠怡、李鎧彤。2020。雲南常見貿易黑塊菌的親緣及分群。台灣林業科學35(1):61-80。

1) 天蕊股份有限公司專案經理，11192 台北市士林區菁山路151號 Project Manager, Tianroei Limited Company. 151 Jingshan Rd, Shihlin Dist., Taipei 11192, Taiwan.

2) 林業試驗所森林保護組副研究員，10066臺北市南海路53號 Associate Researcher, Division of Forest Protection, Taiwan Forestry Research Institute, 53 Nanhai Rd., Taipei 10066, Taiwan.

3) 林業試驗所技術服務組助理研究員，10066 臺北市中正區南海路60號 Assistant Researcher, Technical Service Division, Taiwan Forestry Research Institute, 60 Nanhai Rd., Taipei 10066, Taiwan.

4) 國立臺灣大學森林環境暨資源學系碩士研究生，10617台北市羅斯福路四段1號 Master's student of Department of Forestry and Resource Conservation, National Taiwan University. 1 Roosevelt Road, Section 4, Taipei 10617, Taiwan.

5) 與林有限公司經理人，臺北市中山區南京東路三段201號3樓 Manager, Yulin Limited Company. 3rd Fl., 201 Nanjing East Road, Section 3, Zhongshan District, Taipei 10491, Taiwan.

6) 通訊作者 Corresponding author, e-mail:[email protected]

2018年12月送審 2020年3月通過 Received December 2019, Accepted March 2020.

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Research paper

Phylogenetic Clusters of Commercial Blackish Tuber Species from Yunnan, China

King-Fai Wong,¹⁾ Chuen-Hsu Fu,²⁾ Chieh-Lung Lin,³⁾ Wing-Yi Cheung,⁴⁾ Hoi-Tung Li^5,6)

【Summary】

Morphological and phylogenetic analyses were individually conducted for each commercial blackish Tuber from Yunnan Province, China. An internal transcribed spacer (ITS) phylogenetic analysis of Tuber ascocarps suggests that 3 phylogenetic clusters exist in Yunnan blackish truf- fles, which are the Indicum cluster, Formosanum cluster, and Pseudoexcavatum cluster. In total, 294 ITS sequences were obtained in 2017, among which 76% belonged to the Indicum cluster, 23% belonged to the Formosanum cluster, and 1% belonged to the Pseudoexcavatum cluster.

Among the 10 ITS sequences obtained in 2016, 80% belonged to the Indicum cluster, and 20%

belonged to the Formosanum cluster. The ratio of Indicum clusters to Formosanum clusters was approximately 4:1. Tuber ascocarps of the Pseudoexcavatum cluster were from Huize County;

its members had a distinct morphology and could readily be distinguished from the others. Tuber ascocarps of Indicum and Formosanum clusters had no significant morphological differences, but a pairwise comparison suggested that their ITS sequences only shared 90.56~93.78% simi- larities. Blackish Tuber specimens from Gaoligong Mountain had a good aroma and were cov- ered with soil containing metallic fragments; 75% of Tuber specimens from Gaoligong Moun- tain belonged to the Formosanum cluster.

Key words: black truffles, ITS phylogenetic analysis, morphology, pairwise comparison, phyloge- netic clusters.

Wong KF, Fu CH, Lin CL, Cheung WY, Li HT. 2010. Phylogenetic Clusters of Commercial Blackish Tuber Species from Yunnan, China. Taiwan J For Sci 35(1):61-80.

前言

中國雲南省出產之黑塊菌以印度塊菌 (Tuber indicum Cooke & Massee)為主要貿易 種類，另外也常混入少量的擬外孔塊菌( T.

pseudoexcavatum Y. Wang, G. Moreno, Riousset, Manjón & G. Riousset)。印度塊菌於1892年首先發現於印度的喜瑪拉雅山脈(Cooke and Massee 1892)，於1992年在中國境內也被發現並且產量豐富，現今主要產於中國西部的四川省及雲南省一帶(Zhang et al. 2005, Wang et al. 2006,

Wang and Liu 2009)。印度塊菌在外觀形態上與法國黑孢塊菌(T. melanosporum Vittad.)有極高之 相似度，基因分析上屬同一基因分群，有密切的親緣關係，唯法國黑孢塊菌之氣味比印度塊菌濃郁，以及子囊果外皮、孢子囊內含的孢子數量及孢子紋飾等微細特徵上有些差異(Bonito et al. 2010, 2011, García-Montero et al. 2010, Zambonelli et al. 2016)，要分辨此兩種塊菌並不容易，得靠有經驗的專家以顯微特徵或甚至需

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要分子生物技術輔助，才能鑑定及辨別，有不法商人混入印度塊菌出口，充當高價的法國黑孢塊菌販賣，亦有歐洲的法國黑孢塊菌園受到印度塊菌入侵及污染(Bonito et al. 2011)。

臺灣目前有兩個黑塊菌的發表記錄，皆由退休臺大教授胡弘道所發表，分別為1992年的臺灣塊菌(T. formosanum Hu)及2009年發表的屑塊 菌(T. furfuraceum Hu & Wang) (Hu 1992, Huang et al. 2009)，胡弘道於1996年發表以人工接種方式成功產出臺灣塊菌子實體，說明臺灣本土之氣候環境及殼斗科之青剛櫟適合培育塊菌，然而產量非常少(Hu et al. 2005)。國外研究指出印度塊菌寄主廣泛、不少樹種都能與之共生並形成菌根，臺灣的相關研究也指出印度塊菌能與青剛櫟 (Cyclobalanopsis glauca (Thunb.) Oerst.)小苗共生 並形生菌根，而且菌根形成率較臺灣塊菌佳(Lin 2005)，因此本研究團隊選擇印度塊菌作為接種試驗，初步研究印度塊菌人工栽培的可行性。

本研究團隊早期利用雲南省出產之印度塊菌製作成孢子懸浮液接種青剛櫟小苗，成功形成菌根形態，後來嘗試利用Internal transcribed spacer (ITS) 分子技術鑑定菌根之菌種，結果卻發現小苗所形成之菌根為臺灣塊菌菌根，卻沒有印度塊菌的菌根被發現，同時形成菌根化之菌根大約佔整體接種苗木的22%，因此開始懷疑雲南省的黑塊菌在基因上可能非單一族群。中國雲南省販買之黑塊菌，自多個產區收集混售，寄主植物不確定，而且販買時都是帶土的狀態，難以準確觀察到外皮疣突特徵，亦缺乏鏡檢觀察顯微特徵，主要依據塊菌盤商及市場銷售商的民間經驗而「瞎子摸象」的鑑定，因此極有可能有混合其他塊菌種類。本實驗以大批的雲南黑塊菌作研究，以每顆塊菌為研究單位個別取樣、進行形態及分子鑑定，詳細調查當中之親緣關係，以了解雲南常見貿易黑塊菌的分群，並留下資料後續追蹤接種小苗的確實塊菌種類。

材料與方法

一、塊菌材料

實驗之塊菌子實體於2015~2017年購自中

國雲南省，產季為9-10月，子實體大部分產自會澤縣，產區海拔2300~2400 m，以華山松為主要寄主植物，根據商家鑑定為印度塊菌及擬外孔塊菌；另外亦有購自昆明市木水花野生菌交易市場者，根據商家鑑定及外觀初判有印度塊菌，「中國的夏塊菌」及來自高黎貢山的、香氣特殊及表覆金屬光澤的「夏塊菌」。

二、形態鑑定方法

每顆塊菌子實體以火滅菌過之手術刀切開一角，製作切片以觀察其孢子成熟程度，初步篩選出孢子成熟度大於一半之子實體才進行仔細鑑定，每顆塊菌獨立量度其子實體的直徑長寬、重量、顏色、蟲洞孔洞、軟硬及成熟程度等基本資料後，配以編號，並於攝影箱中拍攝其不同角度之表皮和疣突特徵；接著把子實體剖半，記錄果肉的顏色、內部腔洞的形態及曝露於空氣後氧化的顏色變化等。其後切片加入乳酚溶液製成玻片封存，於Leica DLMB光學顯微鏡下觀察子實體的果皮、子囊及子囊孢子的形態、顏色及紋飾等顯微特徵，拍照記錄後配合Pixitetre 5.8軟體測量子囊孢子大小(n ≥ 100)，作為鑑定依據。

三、分子生物鑑定方法

(一)核酸萃取(DNA extraction)

切一小塊塊菌子實體的菌肉移置離心管內，加lysis buffer後使用研磨棒徹底磨碎，

磨碎後按照產品廠商( 台灣圓點奈米技術股份有限公司)的說明書指引，利用TANBead®

Nucleic Acid Extraction Kit Fungal DNA Auto Plate/Auto Tube，配合萃取機器Smart LabAssist-32 (64)的L-BNA-PK-AUTO Program 萃取DNA，萃取完成之DNA樣本即可繼續PCR 程序，或保存於-30℃冰箱。

(二) 聚合酶鏈鎖反應(polymerase chain reaction;

PCR)及電泳分析

本實驗以I T S 4 及 5 通用性引子對 ( 5 ’ - T C C T C C G C T TAT T G ATAT G C - 3 ’ 及 5’-GGAAGTAAAAGTCGTAACAAGG-3’) (White

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et al. 1990)進行PCR，混合2.5 μL DNA萃取樣本、12.5 μL 2xTaq Master Mix (基龍米克斯生物科技股份有限公司)、各0.5 μL 10 μM引子ITS4/5 及8 μL ddH2O，以配製總容量為24 μL之PCR反應溶液。PCR是用Multigene Thermal Cycler (Labnet International)進行，起始變性(denaturation)溫度為 94℃ 3 min；變性溫度為94℃ 30s，黏合(annealing) 溫度為56℃ 30s，增幅溫度72℃ 30s，子實體樣本循環30週期、菌根樣本循環40週期；最後延展 (extension)溫度72℃ 5 min。經過PCR增幅的DNA 樣本，以含1.4% agarose、0.5 M Tris-acetate-EDTA (TAE)溶液之洋菜膠及5 µL/100 mL Healthview™

nucleic acid stain，於 100 V電壓下進行電泳分析約 25 min。電泳完成後，以Multilmage™ light cabinet (Alphalmager 2200)照相，比較DNA ladder與所得之DNA樣本，以估計各樣本之長度(bp)，然後把產物送至昕穎生醫技術股份有限公司進行定序。

(三)與基因資料庫之比對佐證及親緣樹分析取得定序結果後，與美國生物技術資訊中心網站(NCBI)之資料庫內登錄的基因序列，以核酸基因庫序列群組分析程式(BLAST)作序列相似度比對，綜合比對相似度後得出最相似及具參考價值的佐證結果。由於對NCBI資料庫內印度塊菌序列的確定性存疑，所以使用2015~2016 年經形態記錄之塊菌子實體、純培養菌絲及菌根之序列作基因親緣樹分析，另外再從結果分群(clusters)中各取代表，與2015年在臺灣南投縣東埔山區找到的臺灣塊菌子實體及分離純培養得到的菌絲的序列，以及GenBank中其他序列作基因親緣樹分析，各ITS序列以網路程式MAFFT 7 (Katoh and Standley 2013)對齊後，再以網路程式 Gblocks 0.91b (Castresana 2000)檢查及去掉排列不佳的序列片段，然後以MEGA 6.0 (Tamura et al. 2013)的最大似然分析(ML)作基因親緣分析，

Bootstrap值設定為1000，最後以FigTree 1.4.3 (Rambaut 2014)整合出基因親緣關係圖。

(四)序列成對比較分析(pairwise comparisons) 從基因親緣樹分析的結果分群中各取代表，以網路程式MAFFT 7對齊後，再以網路程

式Gblocks 0.91b檢查及去掉排列不佳的序列片段，然後以PHYLIP 3.695 (Felsensyein 2005) 的DNADIST作成對比較分析，以計算各序列之相似度(similarities)。

結果

一、分子鑑定結果

本研究中得到294條雲南黑塊菌的ITS序列 (accession no.: LC415613~ LC415904, LC419993, LC419994) (Table 1)利用MEGABLAST與 GenBank比對，發現21條序列(7%)與日本發表近似臺灣塊菌及喜馬拉雅塊菌之序列(Kinoshita et al. 2001, 2018)，相似度達97~100%，亦與臺灣塊菌之模式標本序列JN655530 (Qiao et al. 2013) 相似度達99%；另外271條序列(92%)與印度塊菌之序列相似度達96~99%；最後有2條序列(1%)與擬外孔塊菌之序列相似度達97%，與形態鑑定的兩顆擬外孔塊菌(編號YNB17102及YNB17107) 結果吻合。

294條ITS序列加入2015年雲南黑塊菌中抽取4顆雲南黑塊菌的序列(2015YNB1~4)、2016 年10顆塊菌的序列(2016YNB161~YNB1610)、

從臺灣塊菌子實體中分離純培養得到的2個菌絲樣本的序列(T. formosanum_culture S1/S2)及從接種塊菌的青剛櫟小苗中得到的臺灣塊菌菌根之序列(T. formosanum_mycorrhizae)，共311 條序列作ITS親緣關係分析，311條序列排列後序列長947 bp，以Gblocks 0.91b檢查及去掉排列不佳的序列片段後，序列長466 bp。親緣分析結果顯示雲南黑塊菌有3個主要分群，配合形態鑑定及GenBank比對佐證，2個擬外孔塊菌序列以高支持度(BS = 1.00)歸到擬外孔塊菌分群；其餘序列分為兩分群，與臺灣塊菌純培養絲樣本及臺灣塊菌菌根之序列歸到同一分群的為臺灣塊菌分群(BS = 0.62)，另外為印度塊菌分群(BS = 0.79)。2016及2017兩年得到的印度塊菌分群及臺灣塊菌分群比例相約，接近4:1；

2017年的294條黑塊菌序列當中，有223條序列 (76%)屬於印度塊菌分群，69條序列(23%)屬於

臺灣塊菌分群，2條序列(1%)屬於擬外孔塊菌

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分群；2016年黑塊菌序列當中，8條序列(80%) 屬於印度塊菌分群，2條序列(20%)屬於臺灣塊

菌分群；2015年4條黑塊菌序列全屬於印度塊菌分群。

Table 1. List of internal transcribed spacer sequences deposited into GenBank in this study Taxon Origin Isolated from Voucher no. GenBank

accession no.

Tuber formosanum Gaoligong Mt., China Ascocarp YNB17001 LC415613

T. formosanum Gaoligong Mt., China Ascocarp YNB17002 LC415614

T. formosanum Gaoligong Mt., China Ascocarp YNB17006 LC415616 T. formosanum Gaoligong Mt., China Ascocarp YNB17008 LC415617 T. formosanum Gaoligong Mt., China Ascocarp YNB17010 LC415618 T. formosanum Gaoligong Mt., China Ascocarp YNB17011 LC415619 T. formosanum Gaoligong Mt., China Ascocarp YNB17012 LC415620 T. formosanum Gaoligong Mt., China Ascocarp YNB17013 LC415621 T. formosanum Gaoligong Mt., China Ascocarp YNB17014 LC415622 T. formosanum Gaoligong Mt., China Ascocarp YNB17015 LC415623 T. formosanum Gaoligong Mt., China Ascocarp YNB17016 LC415624 T. formosanum Gaoligong Mt., China Ascocarp YNB17018 LC415625 T. formosanum Gaoligong Mt., China Ascocarp YNB17019 LC415626 T. formosanum Huize, Yunnan, China Ascocarp YNB17033 LC415627 T. formosanum Huize, Yunnan, China Ascocarp YNB17039 LC415628 T. formosanum Huize, Yunnan, China Ascocarp YNB17042 LC415629 T. formosanum Huize, Yunnan, China Ascocarp YNB17135 LC415630 T. formosanum Huize, Yunnan, China Ascocarp YNB17141 LC415631

T. formosanum Huize, Yunnan, China Ascocarp YNB17145 LC415632

T. formosanum Huize, Yunnan, China Ascocarp YNB17154 LC415633 T. formosanum Huize, Yunnan, China Ascocarp YNB17155 LC415634 T. formosanum Huize, Yunnan, China Ascocarp YNB17157 LC415635 T. formosanum Huize, Yunnan, China Ascocarp YNB17162 LC415636 T. formosanum Huize, Yunnan, China Ascocarp YNB17163 LC415637 T. formosanum Huize, Yunnan, China Ascocarp YNB17170 LC415638 T. formosanum Huize, Yunnan, China Ascocarp YNB17181 LC415639 T. formosanum Huize, Yunnan, China Ascocarp YNB17183 LC415640 T. formosanum Huize, Yunnan, China Ascocarp YNB17185 LC415641 T. formosanum Huize, Yunnan, China Ascocarp YNB17186 LC415642 T. formosanum Huize, Yunnan, China Ascocarp YNB17192 LC415643 T. formosanum Huize, Yunnan, China Ascocarp YNB17193 LC415644 T. formosanum Gaoligong Mt., China Ascocarp YNB17202 LC415645 T. formosanum Gaoligong Mt., China Ascocarp YNB17203 LC415646

T. formosanum Gaoligong Mt., China Ascocarp YNB17207 LC415649 T. formosanum Gaoligong Mt., China Ascocarp YNB17208 LC415650 T. formosanum Gaoligong Mt., China Ascocarp YNB17209 LC415651

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Table 1 (continue)

T. formosanum Gaoligong Mt., China Ascocarp YNB17210 LC415652 T. formosanum Gaoligong Mt., China Ascocarp YNB17211 LC415653 T. formosanum Gaoligong Mt., China Ascocarp YNB17213 LC415654 T. formosanum Gaoligong Mt., China Ascocarp YNB17215 LC415655

T. formosanum Gaoligong Mt., China Ascocarp YNB17219 LC415657 T. formosanum Gaoligong Mt., China Ascocarp YNB17221 LC415658

T. formosanum Gaoligong Mt., China Ascocarp YNB17224 LC415660 T. formosanum Gaoligong Mt., China Ascocarp YNB17225 LC415661 T. formosanum Gaoligong Mt., China Ascocarp YNB17226 LC415662 T. formosanum Gaoligong Mt., China Ascocarp YNB17227 LC415663 T. formosanum Gaoligong Mt., China Ascocarp YNB17228 LC415664 T. formosanum Gaoligong Mt., China Ascocarp YNB17230 LC415665 T. formosanum Gaoligong Mt., China Ascocarp YNB17231 LC415666 T. formosanum Gaoligong Mt., China Ascocarp YNB17232 LC415667 T. formosanum Gaoligong Mt., China Ascocarp YNB17233 LC415668 T. formosanum Gaoligong Mt., China Ascocarp YNB17234 LC415669 T. formosanum Gaoligong Mt., China Ascocarp YNB17235 LC415670 T. formosanum Gaoligong Mt., China Ascocarp YNB17236 LC415671 T. formosanum Gaoligong Mt., China Ascocarp YNB17237 LC415672 T. formosanum Gaoligong Mt., China Ascocarp YNB17239 LC415673 T. formosanum Gaoligong Mt., China Ascocarp YNB17240 LC415674 T. formosanum Gaoligong Mt., China Ascocarp YNB17243 LC415675 T. formosanum Huize, Yunnan, China Ascocarp YNB17281 LC415676 T. formosanum Huize, Yunnan, China Ascocarp YNB17287 LC415677 T. formosanum Huize, Yunnan, China Ascocarp YNB17290 LC415678 T. formosanum Huize, Yunnan, China Ascocarp YNB17303 LC415679 T. formosanum Huize, Yunnan, China Ascocarp YNB17310 LC415680 T. formosanum Huize, Yunnan, China Ascocarp YNB17316 LC415681

T. formosanum Dung-Pu, Nantou, Taiwan Pure culture S1 LC437961

T. formosanum Dung-Pu, Nantou, Taiwan Pure culture S2 LC437962 T. formosanum Dung-Pu, Nantou, Taiwan Ascocarp TF1 LC437963 T. indicum Gaoligong Mt., China Ascocarp YNB17003 LC415682 T. indicum Gaoligong Mt., China Ascocarp YNB17005 LC415683 T. indicum Gaoligong Mt., China Ascocarp YNB17007 LC415684 T. indicum Gaoligong Mt., China Ascocarp YNB17009 LC415685 T. indicum Gaoligong Mt., China Ascocarp YNB17017 LC415686 T. indicum Huize, Yunnan, China Ascocarp YNB17020 LC415687 T. indicum Huize, Yunnan, China Ascocarp YNB17021 LC415688 T. indicum Huize, Yunnan, China Ascocarp YNB17022 LC415689 T. indicum Huize, Yunnan, China Ascocarp YNB17023 LC415690 T. indicum Huize, Yunnan, China Ascocarp YNB17024 LC415691 T. indicum Huize, Yunnan, China Ascocarp YNB17025 LC415692

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Table 1 (continue)

T. indicum Huize, Yunnan, China Ascocarp YNB17026 LC415693 T. indicum Huize, Yunnan, China Ascocarp YNB17027 LC415694

T. indicum Huize, Yunnan, China Ascocarp YNB17028 LC415695

T. indicum Huize, Yunnan, China Ascocarp YNB17030 LC415697 T. indicum Huize, Yunnan, China Ascocarp YNB17031 LC415698 T. indicum Huize, Yunnan, China Ascocarp YNB17032 LC415699 T. indicum Huize, Yunnan, China Ascocarp YNB17034 LC415700 T. indicum Huize, Yunnan, China Ascocarp YNB17035 LC415701 T. indicum Huize, Yunnan, China Ascocarp YNB17036 LC415702 T. indicum Huize, Yunnan, China Ascocarp YNB17037 LC415703

T. indicum Huize, Yunnan, China Ascocarp YNB17040 LC415705 T. indicum Huize, Yunnan, China Ascocarp YNB17041 LC415706 T. indicum Huize, Yunnan, China Ascocarp YNB17044 LC415707 T. indicum Huize, Yunnan, China Ascocarp YNB17048 LC415708 T. indicum Huize, Yunnan, China Ascocarp YNB17049 LC415709 T. indicum Huize, Yunnan, China Ascocarp YNB17050 LC415710 T. indicum Huize, Yunnan, China Ascocarp YNB17051 LC415711 T. indicum Huize, Yunnan, China Ascocarp YNB17052 LC415712 T. indicum Huize, Yunnan, China Ascocarp YNB17053 LC415713 T. indicum Huize, Yunnan, China Ascocarp YNB17056 LC415714 T. indicum Huize, Yunnan, China Ascocarp YNB17061 LC415715 T. indicum Huize, Yunnan, China Ascocarp YNB17062 LC415716 T. indicum Huize, Yunnan, China Ascocarp YNB17063 LC415717 T. indicum Huize, Yunnan, China Ascocarp YNB17064 LC415718 T. indicum Huize, Yunnan, China Ascocarp YNB17065 LC415719 T. indicum Huize, Yunnan, China Ascocarp YNB17067 LC415720 T. indicum Huize, Yunnan, China Ascocarp YNB17068 LC415721 T. indicum Huize, Yunnan, China Ascocarp YNB17070 LC415722 T. indicum Huize, Yunnan, China Ascocarp YNB17072 LC415723 T. indicum Yunnan market, China Ascocarp YNB17075 LC415724 T. indicum Yunnan market, China Ascocarp YNB17076 LC415725 T. indicum Yunnan market, China Ascocarp YNB17077 LC415726 T. indicum Yunnan market, China Ascocarp YNB17078 LC415727 T. indicum Yunnan market, China Ascocarp YNB17079 LC415728 T. indicum Yunnan market, China Ascocarp YNB17080 LC415729 T. indicum Yunnan market, China Ascocarp YNB17081 LC415730 T. indicum Yunnan market, China Ascocarp YNB17082 LC415731 T. indicum Yunnan market, China Ascocarp YNB17083 LC415732 T. indicum Yunnan market, China Ascocarp YNB17084 LC415733 T. indicum Yunnan market, China Ascocarp YNB17086 LC415734 T. indicum Yunnan market, China Ascocarp YNB17087 LC415735 T. indicum Yunnan market, China Ascocarp YNB17088 LC415736

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Table 1 (continue)

T. indicum Yunnan market, China Ascocarp YNB17089 LC415737 T. indicum Yunnan market, China Ascocarp YNB17090 LC415738

T. indicum Yunnan market, China Ascocarp YNB17091 LC415739

T. indicum Yunnan market, China Ascocarp YNB17093 LC415741 T. indicum Yunnan market, China Ascocarp YNB17094 LC415742 T. indicum Yunnan market, China Ascocarp YNB17095 LC415743 T. indicum Yunnan market, China Ascocarp YNB17096 LC415744 T. indicum Yunnan market, China Ascocarp YNB17097 LC415745 T. indicum Yunnan market, China Ascocarp YNB17098 LC415746 T. indicum Yunnan market, China Ascocarp YNB17099 LC415747 T. indicum Yunnan market, China Ascocarp YNB17100 LC415748 T. indicum Yunnan market, China Ascocarp YNB17101 LC415749 T. indicum Huize, Yunnan, China Ascocarp YNB17103 LC415750 T. indicum Huize, Yunnan, China Ascocarp YNB17104 LC415751 T. indicum Huize, Yunnan, China Ascocarp YNB17105 LC415752 T. indicum Huize, Yunnan, China Ascocarp YNB17106 LC415753 T. indicum Huize, Yunnan, China Ascocarp YNB17108 LC415754 T. indicum Huize, Yunnan, China Ascocarp YNB17109 LC415755 T. indicum Huize, Yunnan, China Ascocarp YNB17110 LC415756 T. indicum Huize, Yunnan, China Ascocarp YNB17111 LC415757 T. indicum Huize, Yunnan, China Ascocarp YNB17112 LC415758 T. indicum Huize, Yunnan, China Ascocarp YNB17113 LC415759 T. indicum Huize, Yunnan, China Ascocarp YNB17114 LC415760 T. indicum Huize, Yunnan, China Ascocarp YNB17115 LC415761 T. indicum Huize, Yunnan, China Ascocarp YNB17116 LC415762 T. indicum Huize, Yunnan, China Ascocarp YNB17117 LC415763 T. indicum Huize, Yunnan, China Ascocarp YNB17118 LC415764 T. indicum Huize, Yunnan, China Ascocarp YNB17119 LC415765 T. indicum Huize, Yunnan, China Ascocarp YNB17120 LC415766 T. indicum Huize, Yunnan, China Ascocarp YNB17121 LC415767 T. indicum Huize, Yunnan, China Ascocarp YNB17122 LC415768 T. indicum Huize, Yunnan, China Ascocarp YNB17123 LC415769 T. indicum Huize, Yunnan, China Ascocarp YNB17124 LC415770 T. indicum Huize, Yunnan, China Ascocarp YNB17125 LC415771 T. indicum Huize, Yunnan, China Ascocarp YNB17126 LC415772 T. indicum Huize, Yunnan, China Ascocarp YNB17127 LC415773 T. indicum Huize, Yunnan, China Ascocarp YNB17128 LC415774 T. indicum Huize, Yunnan, China Ascocarp YNB17129 LC415775

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Table 1 (continue)

T. indicum Huize, Yunnan, China Ascocarp YNB17140 LC415785 T. indicum Huize, Yunnan, China Ascocarp YNB17142 LC415786 T. indicum Huize, Yunnan, China Ascocarp YNB17143 LC415787 T. indicum Huize, Yunnan, China Ascocarp YNB17144 LC415788 T. indicum Huize, Yunnan, China Ascocarp YNB17146 LC415789 T. indicum Huize, Yunnan, China Ascocarp YNB17147 LC415790 T. indicum Huize, Yunnan, China Ascocarp YNB17148 LC415791 T. indicum Huize, Yunnan, China Ascocarp YNB17149 LC415792 T. indicum Huize, Yunnan, China Ascocarp YNB17150 LC415793 T. indicum Huize, Yunnan, China Ascocarp YNB17151 LC415794 T. indicum Huize, Yunnan, China Ascocarp YNB17152 LC415795 T. indicum Huize, Yunnan, China Ascocarp YNB17153 LC415796 T. indicum Huize, Yunnan, China Ascocarp YNB17156 LC415797 T. indicum Huize, Yunnan, China Ascocarp YNB17158 LC415798 T. indicum Huize, Yunnan, China Ascocarp YNB17159 LC415799 T. indicum Huize, Yunnan, China Ascocarp YNB17160 LC415800

T. indicum Huize, Yunnan, China Ascocarp YNB17164 LC415802 T. indicum Huize, Yunnan, China Ascocarp YNB17165 LC415803 T. indicum Huize, Yunnan, China Ascocarp YNB17166 LC415804 T. indicum Huize, Yunnan, China Ascocarp YNB17167 LC415805 T. indicum Huize, Yunnan, China Ascocarp YNB17168 LC415806 T. indicum Huize, Yunnan, China Ascocarp YNB17169 LC415807 T. indicum Huize, Yunnan, China Ascocarp YNB17171 LC415808 T. indicum Huize, Yunnan, China Ascocarp YNB17172 LC415809 T. indicum Huize, Yunnan, China Ascocarp YNB17173 LC415810 T. indicum Huize, Yunnan, China Ascocarp YNB17175 LC415811 T. indicum Huize, Yunnan, China Ascocarp YNB17176 LC415812 T. indicum Huize, Yunnan, China Ascocarp YNB17177 LC415813 T. indicum Huize, Yunnan, China Ascocarp YNB17178 LC415814 T. indicum Huize, Yunnan, China Ascocarp YNB17179 LC415815 T. indicum Huize, Yunnan, China Ascocarp YNB17180 LC415816 T. indicum Huize, Yunnan, China Ascocarp YNB17182 LC415817 T. indicum Huize, Yunnan, China Ascocarp YNB17184 LC415818 T. indicum Huize, Yunnan, China Ascocarp YNB17187 LC415819 T. indicum Huize, Yunnan, China Ascocarp YNB17188 LC415820 T. indicum Huize, Yunnan, China Ascocarp YNB17189 LC415821 T. indicum Huize, Yunnan, China Ascocarp YNB17190 LC415822 T. indicum Huize, Yunnan, China Ascocarp YNB17191 LC415823 T. indicum Huize, Yunnan, China Ascocarp YNB17194 LC415824

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Table 1 (continue)

T. indicum Huize, Yunnan, China Ascocarp YNB17200 LC415830 T. indicum Gaoligong Mt., China Ascocarp YNB17201 LC415831 T. indicum Gaoligong Mt., China Ascocarp YNB17212 LC415832 T. indicum Gaoligong Mt., China Ascocarp YNB17214 LC415833 T. indicum Gaoligong Mt., China Ascocarp YNB17216 LC415834 T. indicum Gaoligong Mt., China Ascocarp YNB17217 LC415835 T. indicum Gaoligong Mt., China Ascocarp YNB17220 LC415836 T. indicum Gaoligong Mt., China Ascocarp YNB17223 LC415837

T.inducum Gaoligong Mt., China Ascocarp YNB17238 LC415838

T. indicum Gaoligong Mt., China Ascocarp YNB17241 LC415839 T. indicum Gaoligong Mt., China Ascocarp YNB17242 LC415840 T. indicum Gaoligong Mt., China Ascocarp YNB17244 LC415841 T. indicum Yunnan market, China Ascocarp YNB17245 LC415842s

T. indicum Yunnan market, China Ascocarp YNB17247 LC415844 T. indicum Yunnan market, China Ascocarp YNB17248 LC415845 T. indicum Yunnan market, China Ascocarp YNB17249 LC415846 T. indicum Yunnan market, China Ascocarp YNB17250 LC415847 T. indicum Yunnan market, China Ascocarp YNB17251 LC415848 T. indicum Yunnan market, China Ascocarp YNB17252 LC415849 T. indicum Yunnan market, China Ascocarp YNB17253 LC415850 T. indicum Yunnan market, China Ascocarp YNB17254 LC415851 T. indicum Yunnan market, China Ascocarp YNB17255 LC415852 T. indicum Yunnan market, China Ascocarp YNB17256 LC415853 T. indicum Yunnan market, China Ascocarp YNB17257 LC415854 T. indicum Yunnan market, China Ascocarp YNB17258 LC415855 T. indicum Yunnan market, China Ascocarp YNB17259 LC415856 T. indicum Yunnan market, China Ascocarp YNB17260 LC415857 T. indicum Yunnan market, China Ascocarp YNB17261 LC415858 T. indicum Yunnan market, China Ascocarp YNB17262 LC415859 T. indicum Yunnan market, China Ascocarp YNB17263 LC415860 T. indicum Huize, Yunnan, China Ascocarp YNB17264 LC415861 T. indicum Huize, Yunnan, China Ascocarp YNB17265 LC415862 T. indicum Huize, Yunnan, China Ascocarp YNB17266 LC415863 T. indicum Huize, Yunnan, China Ascocarp YNB17267 LC415864 T. indicum Huize, Yunnan, China Ascocarp YNB17268 LC415865 T. indicum Huize, Yunnan, China Ascocarp YNB17269 LC415866 T. indicum Huize, Yunnan, China Ascocarp YNB17270 LC415867 T. indicum Huize, Yunnan, China Ascocarp YNB17271 LC415868

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從以上結果的3個分群中隨機各取20個代表序列，與2015年在臺灣南投縣東埔山區找到的臺灣塊菌子實體及其分離純培養得到的菌絲序列(accession no.: LC437961~LC437963)，以及

GenBank中其他序列(Table 2)作基因親緣樹分析圖 (Fig. 1)，62條序列排列後序列長959 bp，

以Gblocks 0.91b檢查及去掉排列不佳的序列片段後，序列長505 bp。結果顯示雲南黑塊菌的 Table 1 (continue)

T. indicum Huize, Yunnan, China Ascocarp YNB17272 LC415869 T. indicum Huize, Yunnan, China Ascocarp YNB17273 LC415870 T. indicum Huize, Yunnan, China Ascocarp YNB17275 LC415871

T. indicum Huize, Yunnan, China Ascocarp YNB17277 LC415873 T. indicum Huize, Yunnan, China Ascocarp YNB17278 LC415874 T. indicum Huize, Yunnan, China Ascocarp YNB17279 LC415875 T. indicum Huize, Yunnan, China Ascocarp YNB17280 LC415876 T. indicum Huize, Yunnan, China Ascocarp YNB17282 LC415877 T. indicum Huize, Yunnan, China Ascocarp YNB17283 LC415878 T. indicum Huize, Yunnan, China Ascocarp YNB17284 LC415879 T. indicum Huize, Yunnan, China Ascocarp YNB17285 LC415880 T. indicum Huize, Yunnan, China Ascocarp YNB17286 LC415881 T. indicum Huize, Yunnan, China Ascocarp YNB17288 LC415882 T. indicum Huize, Yunnan, China Ascocarp YNB17289 LC415883 T. indicum Huize, Yunnan, China Ascocarp YNB17291 LC415884 T. indicum Huize, Yunnan, China Ascocarp YNB17292 LC415885 T. indicum Huize, Yunnan, China Ascocarp YNB17293 LC415886 T. indicum Huize, Yunnan, China Ascocarp YNB17294 LC415887 T. indicum Huize, Yunnan, China Ascocarp YNB17295 LC415888 T. indicum Huize, Yunnan, China Ascocarp YNB17296 LC415889 T. indicum Huize, Yunnan, China Ascocarp YNB17297 LC415890 T. indicum Huize, Yunnan, China Ascocarp YNB17298 LC415891 T. indicum Huize, Yunnan, China Ascocarp YNB17299 LC415892 T. indicum Huize, Yunnan, China Ascocarp YNB17300 LC415893 T. indicum Huize, Yunnan, China Ascocarp YNB17301 LC415894 T. indicum Huize, Yunnan, China Ascocarp YNB17302 LC415895 T. indicum Huize, Yunnan, China Ascocarp YNB17304 LC415896 T. indicum Huize, Yunnan, China Ascocarp YNB17308 LC415897 T. indicum Huize, Yunnan, China Ascocarp YNB17309 LC415898 T. indicum Huize, Yunnan, China Ascocarp YNB17313 LC415899 T. indicum Huize, Yunnan, China Ascocarp YNB17314 LC415900 T. indicum Huize, Yunnan, China Ascocarp YNB17318 LC415901 T. indicum Huize, Yunnan, China Ascocarp YNB17320 LC415902 T. indicum Huize, Yunnan, China Ascocarp YNB17323 LC415903 T. indicum Huize, Yunnan, China Ascocarp YNB17326 LC415904 T. pseudoexcavatum Huize, Yunnan, China Ascocarp YNB17102 LC419993 T. pseudoexcavatum Huize, Yunnan, China Ascocarp YNB17107 LC419994

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3個分群狀況相同，擬外孔塊菌分群的2條序列與GenBank的擬外孔塊菌序列以高支持度(BS = 0.98)分為一群，其他雲南黑塊菌與法國黑孢塊菌為姐妹群(BS = 1.00)，但亦以高支持度(BS = 0.85/0.84)分為兩分群，印度塊菌分群包括了雲

南黑塊菌的20個印度塊菌分群代表，以及4條

GenBank的印度塊菌序列；其他20個臺灣塊菌分群代表、臺灣塊菌子實體及分離純培養得到的菌絲的序列及GenBank的序列分到臺灣塊菌分群。

Table 2. Details of internal transcribed spacer sequences used in the phylogenetic study in Fig. 1

Taxon Origin Voucher no. GenBank

Reference accession no.

Tuber formosanum Gaoligong Mt., China YNB 17001 LC415613 This study T. formosanum Gaoligong Mt., China YNB 17004 LC415615 This study T. formosanum Gaoligong Mt., China YNB 17011 LC415619 This study T. formosanum Gaoligong Mt., China YNB 17015 LC415623 This study T. formosanum Gaoligong Mt., China YNB 17019 LC415626 This study T. formosanum Huize, Yunnan, China YNB 17033 LC415627 This study T. formosanum Huize, Yunnan, China YNB 17042 LC415629 This study T. formosanum Huize, Yunnan, China YNB 17135 LC415630 This study T. formosanum Huize, Yunnan, China YNB 17157 LC415635 This study T. formosanum Huize, Yunnan, China YNB 17170 LC415638 This study T. formosanum Huize, Yunnan, China YNB 17183 LC415640 This study T. formosanum Gaoligong Mt., China YNB 17202 LC415645 This study T. formosanum Gaoligong Mt., China YNB 17222 LC415659 This study T. formosanum Gaoligong Mt., China YNB 17228 LC415664 This study T. formosanum Gaoligong Mt., China YNB 17230 LC415665 This study T. formosanum Gaoligong Mt., China YNB 17243 LC415675 This study T. formosanum Huize, Yunnan, China YNB 17281 LC415676 This study T. formosanum Huize, Yunnan, China YNB 17287 LC415677 This study T. formosanum Huize, Yunnan, China YNB 17290 LC415678 This study

T. formosanum Huize, Yunnan, China YNB 17303 LC415679 This study

T. formosanum Dung-Pu, Nantou, Taiwan S1 LC437961 This study T. formosanum Dung-Pu, Nantou, Taiwan S2 LC437962 This study T. formosanum Dung-Pu, Nantou, Taiwan TF1 LC437963 This study T. formosanum Ho-She, Nantou, Taiwan HKAS62628(holotype) JN655530 Qiao et al. 2013 T. formosanum Ho-She, Nantou, Taiwan T12_HKAS48268 GU979048 Qiao et al. 2013 T. formosanum Dung-Pu, Nantou, Taiwan HU070105C_DungPu03 FJ176919 Huang et al. 2009 T. formosanum Dung-Pu, Nantou, Taiwan HU070105A_DungPu01 FJ176914 Huang et al. 2009

T. himalayense Japan K464 AB553394 Kinoshita et al. 2018

T. himalayense Japan N123 AB553402 Kinoshita et al. 2011

T. himalayense Huize, Yunnan, China HKAS25689 AY773356 Wang et al. 2006 T. indicum Gaoligong Mt., China YNB 17003 LC415682 This study T. indicum Gaoligong Mt., China YNB 17005 LC415683 This study T. indicum Gaoligong Mt., China YNB 17007 LC415684 This study T. indicum Gaoligong Mt., China YNB 17009 LC415685 This study T. indicum Gaoligong Mt., China YNB 17017 LC415686 This study

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各序列經成對比較之相似度列於Table 3，

臺灣塊菌分群內序列相似度為96.73~100%，

印度塊菌分群內序列相似度為97.53~100%，

比較臺灣塊菌及印度塊菌分群之序列相似度為9 0 . 5 6 ~ 9 3 . 7 8 % ，而比較擬外孔塊菌分群與臺灣塊菌及印度塊菌分群之序列相似度為 68.23~74.84%。

二、形態鑑定結果

塊菌子實體材料中，以顯微鏡觀察孢子

成熟程度，篩選出25%為不成熟，無法用作後

續接種材料，所以沒有作形態及分子鑑定，未成熟的塊菌子實體外皮紅棕色，切開菌肉淡橘色，白色菌脈不明顯。成熟的塊菌子實體

大小範圍為17~73×16~59 mm，平均大小為 36.3×30.4 mm，平均重量17.5 g，從外型及表皮特徵看，大部分塊菌子實體外觀相似，未有任何明顯特徵可分辦種類，外皮皆為深褐或黑色，外皮有4至6邊錐型疣突、1 cm有5~6個疣突，外觀合乎印度塊菌或臺灣塊菌之描述；當中只有編號YNB17102及YNB17107明顯不同，

兩顆外皮褐色，疣狀突出比印度塊菌細而淺，

1 cm有16個疣突，以顯微鏡觀察子囊內以5~7個孢子為主，與印度塊菌及臺灣塊菌中每子囊主要2~4個孢子可以明顯區別(Fig. 2)，以形態鑑定為擬外孔塊菌。氣味方面，只有產自高黎貢山產區之塊菌有干貝般的特殊香氣，其他塊菌都不具明顯香味。

Table 2 (continue)

T. indicum Huize, Yunnan, China YNB 17020 LC415687 This study T. indicum Huize, Yunnan, China YNB 17030 LC415697 This study T. indicum Huize, Yunnan, China YNB 17040 LC415705 This study T. indicum Yunnan market, China YNB 17080 LC415729 This study T. indicum Yunnan market, China YNB 17100 LC415748 This study T. indicum Huize, Yunnan, China YNB 17188 LC415820 This study

T. indicum Huize, Yunnan, China YNB 17197 LC415827 This study

T. indicum Gaoligong Mt., China YNB 17214 LC415833 This study T. indicum Gaoligong Mt., China YNB 17217 LC415835 This study T. indicum Gaoligong Mt., China YNB 17220 LC415836 This study T. indicum Gaoligong Mt., China YNB 17238 LC415838 This study T. indicum Gaoligong Mt., China YNB 17244 LC415841 This study T. indicum Yunnan market, China YNB 17249 LC415846 This study T. indicum Yunnan market, China YNB 17252 LC415849 This study T. indicum Huize, Yunnan, China YNB 17293 LC415886 This study

T. indicum Yunnan, China Tind-yn20 DQ375515 Wang et al. 2006

T. indicum Hulli, Sichuan, China Tind-hl04 DQ375497 Wang et al. 2006

T. melanosporum France Tmel-eu05 DQ388876 Wang et al. 2006

T. melanosporum France Tmel-eu08 DQ388877 Wang et al. 2006

T. panzhihuanense Panzhihua, Sichuan, China DXJ267 (holotype) JQ978644 Deng et al. 2012 T. panzhihuanense Kunming, Yunnan, China DXJ260 JQ978648 Deng et al. 2012

T. pseudoexcavatum China Tpse-pzh02 DQ329371 Huang et al. 2009

T. pseudoexcavatum China Tpse-yn05 DQ329374 Huang et al. 2009

T. pseudoexcavatum Yunnan, China YNB 17102 LC419993 This study T. pseudoexcavatum Yunnan, China YNB 17107 LC419994 This study

(14)

Fig. 1. Phylogenetic tree of Yuannan blackish Tuber and related Tuber species of the Melanosporum Clade based on the ITS-rDNA sequences. Tuber panzhihuanense was used as the outgroup taxa. Numbers near branches indicated the bootstrap values (BS) obtained from maximum likelihood (ML) analysis. Values below 50 are not indicated.

(15)

Table 3. Pairwise comparisons of sequences of Indicum, Formosanum, and Pseudoexcavatum clusters of Yunnan blackish Tuber species Note: 1) Similarities are shown in percentages. (1~10) represent Formosanum clusters, (11~20) represent Indicum clusters, and (21 and 22) represent Pseudoexcava-tum clusters.2) The gray area highlights similarities between Formosanum and Indicum clusters.

雲南常見貿易黑塊菌的親緣及分群 黃勁暉

雲南常見貿易黑塊菌的親緣及分群黃勁暉